Method for the diagnosis, prognosis and treatment of prostate cancer metastasis

ABSTRACT

The present invention relates to a method for the diagnosis or the prognosis of metastasis in prostate cancer which comprises determining if the c-MAF gene is amplified in a primary tumor sample. Likewise, the invention also relates to a method for the diagnosis or the prognosis of metastasis in prostate cancer, as well as to a method for determining the tendency to develop bone metastasis with respect to metastasis in other organs, which comprise determining the c-MAF expression level. Finally, the invention relates to the use of a c-MAF inhibitor as therapeutic target for treating the prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/713,318, filed on Oct. 12,2012, and incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The content of the electronically submitted sequence listing(“3190_(—)0030001 SEQIDListing_ascii.txt”, 48,245 bytes, created on Oct.7, 2013) filed with the application is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the diagnosis or the prognosis ofmetastasis in prostate cancer based on determining if the c-MAF gene,within the 16q22-24 genomic region, is amplified in a primary tumorsample. Likewise, the invention also relates to a method for thediagnosis or the prognosis of metastasis in prostate cancer, as well asto a method for designing a customized therapy in a subject withprostate cancer, which comprises determining the c-MAF gene expressionlevel or 16q22-24 amplification. Finally, the invention relates to theuse of a c-MAF inhibitor as a therapeutic target for the treatment ofprostate cancer metastasis.

2. Background Art

The Problem:

Metastasis, a complex process caused by elaborate interactions betweentumor cells and the surrounding normal tissues in different vitalorgans, accounts for 90 percent of all cancer deaths in patients withsolid tumors. The molecular and cellular mechanisms that lead primarytumors to form metastases must be understood in order to better addressthis major life-threatening problem. The identification of metastasisgenes and mechanisms is essential for understanding the basic biology ofthis lethal condition and its implications for clinical practice.

Introduction and Interest: Prostate Organ-Specific Metastasis

Prostate cancer is a form of cancer that develops in the prostate, agland in the male reproductive system. Most prostate cancers are slowgrowing; however, there are cases of aggressive prostate cancers. Thecancer cells may metastasize (spread) from the prostate to other partsof the body, particularly the bones and lymph nodes. Prostate cancer maycause pain, difficulty in urinating, problems during sexual intercourse,or erectile dysfunction. Other symptoms can potentially develop duringlater stages of the disease.

Rates of detection of prostate cancers vary widely across the world,with South and East Asia detecting less frequently than in Europe, andespecially the United States. Prostate cancer tends to develop in menover the age of fifty and although it is one of the most prevalent typesof cancer in men, many never have symptoms, undergo no therapy, andeventually die of other causes. About two-thirds of cases are slowgrowing, the other third more aggressive and fast developing.

Many factors, including genetics and diet, have been implicated in thedevelopment of prostate cancer. The presence of prostate cancer may beindicated by symptoms, physical examination, prostate-specific antigen(PSA), or biopsy. The PSA test increases cancer detection but does notdecrease mortality. Moreover, prostate test screening is controversialat the moment and may lead to unnecessary, even harmful, consequences insome patients. Nonetheless, suspected prostate cancer is typicallyconfirmed by taking a biopsy of the prostate and examining it under amicroscope. Further tests, such as CT scans and bone scans, may beperformed to determine whether prostate cancer has spread.

Management strategies for prostate cancer should be guided by theseverity of the disease. Many low-risk tumors can be safely followedwith active surveillance. Curative treatment generally involves surgery,various forms of radiation therapy, or, less commonly, cryosurgery;hormonal therapy and chemotherapy are generally reserved for cases ofadvanced disease (although hormonal therapy may be given with radiationin some cases).

The age and underlying health of the man, the extent of metastasis,appearance under the microscope and response of the cancer to initialtreatment are important in determining the outcome of the disease. Thedecision whether or not to treat localized prostate cancer (a tumor thatis contained within the prostate) with curative intent is a patienttrade-off between the expected beneficial and harmful effects in termsof patient survival and quality of life.

The specific causes of prostate cancer remain unknown. Geneticbackground may contribute to prostate cancer risk, as suggested byassociations with race, family, and specific gene variants. No singlegene is responsible for prostate cancer; many different genes have beenimplicated. Mutations in BRCA1 and BRCA2, important risk factors forovarian cancer and breast cancer in women, have also been implicated inprostate cancer. Other linked genes include the Hereditary Prostatecancer gene 1 (HPC1), the androgen receptor, and the vitamin D receptor.TMPRSS2-ETS gene family fusion, specifically TMPRSS2-ERG orTMPRSS2-ETV1/4 promotes cancer cell growth.

Loss of cancer suppressor genes, early in the prostatic carcinogenesis,have been localized to chromosomes 8p, 10q, 13q, and 16q. P53 mutationsin the primary prostate cancer are relatively low and are morefrequently seen in metastatic settings, hence, p53 mutations are a lateevent in pathology of prostate cancer. Other tumor suppressor genes thatare thought to play a role in prostate cancer include PTEN (gene) andKAI1. Up to 70 percent of men with prostate cancer have lost one copy ofthe PTEN gene at the time of diagnosis. Relative frequency of loss ofE-cadherin and CD44 has also been observed.

Prostate cancer is classified as an adenocarcinoma, or glandular cancer,that begins when normal semen-secreting prostate gland cells mutate intocancer cells. The region of prostate gland where the adenocarcinoma ismost common is the peripheral zone. Initially, small clumps of cancercells remain confined to otherwise normal prostate glands, a conditionknown as carcinoma in situ or prostatic intraepithelial neoplasia (PIN).Although there is no proof that PIN is a cancer precursor, it is closelyassociated with cancer. Over time, these cancer cells begin to multiplyand spread to the surrounding prostate tissue (the stroma) forming atumor. Eventually, the tumor may grow large enough to invade nearbyorgans such as the seminal vesicles or the rectum, or the tumor cellsmay develop the ability to travel in the bloodstream and lymphaticsystem. Prostate cancer is considered a malignant tumor because it is amass of cells that can invade other parts of the body. This invasion ofother organs is called metastasis. Prostate cancer most commonlymetastasizes to the bones, lymph nodes, and may invade rectum, bladderand lower ureters after local progression.

Molecular Traits of Prostate Cancer

RUNX2 is a transcription factor that prevents cancer cells fromundergoing apoptosis thereby contributing to the development of prostatecancer.

The PI3k/Akt signaling cascade works with the transforming growth factorbeta/SMAD signaling cascade to ensure prostate cancer cell survival andprotection against apoptosis. X-linked inhibitor of apoptosis (XIAP) ishypothesized to promote prostate cancer cell survival and growth and isa target of research because if this inhibitor can be shut down then theapoptosis cascade can carry on its function in preventing cancer cellproliferation. Macrophage inhibitory cytokine-1 (MIC-1) stimulates thefocal adhesion kinase (FAK) signaling pathway which leads to prostatecancer cell growth and survival.

The androgen receptor helps prostate cancer cells to survive and is atarget for many anti-cancer research studies; so far, inhibiting theandrogen receptor has only proven to be effective in mouse studies.Prostate specific membrane antigen (PSMA) stimulates the development ofprostate cancer by increasing folate levels for the cancer cells to useto survive and grow; PSMA increases available folates for use byhydrolyzing glutamated folates.

Diagnosis

The only test that can fully confirm the diagnosis of prostate cancer isa biopsy, the removal of small pieces of the prostate for microscopicexamination. However, prior to a biopsy, less invasive testing can beconducted.

There are also several other tests that can be used to gather moreinformation about the prostate and the urinary tract. Digital rectalexamination (DRE) may allow a doctor to detect prostate abnormalities.Cystoscopy shows the urinary tract from inside the bladder, using athin, flexible camera tube inserted down the urethra. Transrectalultrasonography creates a picture of the prostate using sound waves froma probe in the rectum.

Prostate Imaging

Ultrasound (US) and Magnetic Resonance Imaging (MRI) are the two mainimaging methods used for prostate cancer detection.

Biopsy

Micrograph showing a prostate cancer (conventional adenocarcinoma) withperineural invasion. H&E stain.

If cancer is suspected, a biopsy is offered expediently. During a biopsya urologist or radiologist obtains tissue samples from the prostate viathe rectum. A biopsy gun inserts and removes special hollow-core needles(usually three to six on each side of the prostate) in less than asecond. Prostate biopsies are routinely done on an outpatient basis andrarely require hospitalization. Fifty-five percent of men reportdiscomfort during prostate biopsy.

Gleason Score

The tissue samples are then examined under a microscope to determinewhether cancer cells are present, and to evaluate the microscopicfeatures (or Gleason score) of any cancer found. Prostate specificmembrane antigen is a transmembrane carboxypeptidase and exhibits folatehydrolase activity. This protein is overexpressed in prostate cancertissues and is associated with a higher Gleason score.

Tumor Markers

Tissue samples can be stained for the presence of PSA and other tumormarkers in order to determine the origin of malignant cells that havemetastasized.

Small cell carcinoma is a very rare (1%) type of prostate cancer thatcannot be diagnosed using the PSA. As of 2009 researchers are trying todetermine the best way to screen for this type of prostate cancerbecause it is a relatively unknown and rare type of prostate cancer butvery serious and quick to spread to other parts of the body. Possiblemethods include chromatographic separation methods by mass spectrometry,or protein capturing by immunoassays or immunized antibodies. The testmethod will involve quantifying the amount of the biomarker PCI, withreference to the Gleason Score. Not only is this test quick, it is alsosensitive. It can detect patients in the diagnostic grey zone,particularly those with a serum free to total Prostate Specific Antigenratio of 10-20%.

The expression of Ki-67 by immunohistochemistry may be a significantpredictor of patient outcome for men with prostate cancer.

Classification

An important part of evaluating prostate cancer is determining thestage, or how far the cancer has spread. Knowing the stage helps defineprognosis and is useful when selecting therapies. The most common systemis the four-stage TNM system (abbreviated from Tumor/Nodes/Metastases).Its components include the size of the tumor, the number of involvedlymph nodes, and the presence of any other metastases.

The most important distinction made by any staging system is whether ornot the cancer is still confined to the prostate. In the TNM system,clinical T1 and T2 cancers are found only in the prostate, while T3 andT4 cancers have spread elsewhere. Several tests can be used to look forevidence of spread. These include computed tomography to evaluate spreadwithin the pelvis, bone scans to look for spread to the bones, andendorectal coil magnetic resonance imaging to closely evaluate theprostatic capsule and the seminal vesicles. Bone scans should revealosteoblastic appearance due to increased bone density in the areas ofbone metastasis—opposite to what is found in many other cancers thatmetastasize.

After a prostate biopsy, a pathologist looks at the samples under amicroscope. If cancer is present, the pathologist reports the grade ofthe tumor. The grade tells how much the tumor tissue differs from normalprostate tissue and suggests how fast the tumor is likely to grow. TheGleason system is used to grade prostate tumors from 2 to 10, where aGleason score of 10 indicates the most abnormalities. The pathologistassigns a number from 1 to 5 for the most common pattern observed underthe microscope, then does the same for the second-most-common pattern.The sum of these two numbers is the Gleason score. The Whitmore-Jewettstage is another method sometimes used.

Screening

Prostate cancer screening is an attempt to find unsuspected cancers, andmay lead to more specific follow-up tests such as a biopsy, with cellsamples taken for closer study. Options include the digital rectal exam(DRE) and the prostate-specific antigen (PSA) blood test. Such screeningis controversial and, in some patients, may lead to unnecessary, evenharmful, consequences. A 2010 analysis concluded that routine screeningwith either a DRE or PSA is not supported by the evidence as there is nomortality benefit from screening. More recently, the United StatesPreventive Services Task Force (USPSTF) recommended against the PSA testfor prostate cancer screening in healthy men. This USPSTFrecommendation, released in October 2011, is based on “review ofevidence” studies concluding that “Prostate-specific antigen-basedscreening results in small or no reduction in prostate cancer-specificmortality and is associated with harms related to subsequent evaluationand treatments, some of which may be unnecessary.

Modern screening tests have found cancers that might never havedeveloped into serious disease, and that “the slight reduction of riskby surgically removing the prostate or treating it with radiation maynot outweigh the substantial side effects of these treatments,” anopinion also shared by the CDC.

Aggressive Cancer

If the cancer has spread beyond the prostate, treatment optionssignificantly change, so most doctors that treat prostate cancer use avariety of nomograms to predict the probability of spread. Treatment bywatchful waiting/active surveillance, external beam radiation therapy,brachytherapy, cryosurgery, HIFU, and surgery are, in general, offeredto men whose cancer remains within the prostate. Hormonal therapy andchemotherapy are often reserved for disease that has spread beyond theprostate. However, there are exceptions: radiation therapy may be usedfor some advanced tumors, and hormonal therapy is used for some earlystage tumors. Cryotherapy (the process of freezing the tumor), hormonaltherapy, and chemotherapy may also be offered if initial treatment failsand the cancer progresses.

If the disease has reached clinical stage T3 or T4, it is classified asadvanced prostate cancer. Advanced prostate cancer with bone metastasisor lymph node metastasis is more likely to cause Prostate CancerSymptoms than is an early stage of the disease. Doctors usually checkfor bone metastasis and lymph node metastasis which are denotedrespectively by M and N in clinical staging.

In clinical stage T3, the tumor has extended beyond the prostaticcapsule, possibly into the seminal vesicles, and is specifically calledextraprostatic extension. Extraprostatic means “independent of theprostate gland.” In clinical stage T4, the disease invades surroundingorgans (other than the seminal vesicles) such as the bladder neck,external sphincter, or rectum.

Metastasis is more likely to occur during advanced prostate cancer.Metastatic disease refers to prostate cancer that has left the prostategland and its neighboring organs. Advanced prostate cancer bonemetastasis and lymph node metastasis, which can be local or distant, areboth associated with advanced prostate cancer. Metastases may involvesymptoms that are not in the Prostate Cancer Treatment Guide.

Prostate Cancer Lymph Node Metastasis

The body produces a fluid called lymph which contains white blood cellsand circulates through the lymphatic system. Lymph nodes are small ovalor circular organs that filter this fluid. Cancerous cells thatcirculate through the body can become trapped in the lymph nodes. Oncetrapped, cancerous cells can begin their cycle of unhealthy division andresult in lymph node metastasis.

There are two types of lymph node metastasis: local and distant. Locallymph node metastasis is designated by clinical stage N1. Two lymphnodes lie on either side of the bladder. Because these nodes are closeto the prostate gland, metastasis is considered local. If cancerouscells begin to grow in any other lymph node, the metastasis isconsidered distant. Distant lymph node metastasis is denoted by clinicalstage M1a.

Prostate Cancer Bone Matastasis

Primary cases of bone cancer are relatively rare. Patients who developbone cancer are more likely to develop the disease as a result ofadvanced prostate cancer metastasis. In prostate cancer, extensionleading to bone disease is designated by a clinical stage M1b. If aperson develops bone disease as a result of prostate cancer, he does notnow have bone cancer. Because the cancer is classified according towhere it originated, he has prostate cancer with bone metastasis.

Skeletal metastases occur in more than 80% of advanced-stage prostatecancer and they confer a high level of morbidity, a 5-year survival rateof 25% and median survival of approximately 40 months. Of the estimatedone million annual deaths associated with metastatic bone disease in theUSA, EU and Japan, approximately 20% are cases of advanced-stageprostate cancer. Treatment-naïve metastatic prostate cancer is largelysensitive to androgen-deprivation therapy but progression tocastration-resistant prostate cancer occurs 18-20 months after startingtreatment. Metastatic bone disease causes some of the most distressingsymptoms of advanced-stage cancer; estimates indicate that treatment ofbone pain is required in approximately 30% of men with castrationresistant prostate cancer and associated with metastatic bone disease;with 22% requiring treatment for singular or multiple pathologicalskeletal fractures; 7% for spinal-cord compression; 3-4% for hemiparesisor paresis. At first diagnosis of bone metastasis disease therapeuticintervention will usually involve systemic chemotherapy, hormonaltherapy and bisphosphonates or Denosumab, which are mostly palliativeoptions with the intention of reducing pain.

In healthy skeletal bone, an equal balance of new bone matrix formationand old bone matrix resorption is achieved via coordinated activity ofbone-degrading osteoclasts and bone-forming osteoblasts. Duringmetastasis bone disease, the normal balance of bone resorption andformation is disrupted by the homotypic and heterotypic cell-cellinteractions that occur between invading tumor cells, osteoblasts andostoclasts. Most patients with secondary bone tumors—including thoseassociated with castration resistant prostate cancer-present withosteolytic lesions. Therefore, most treatment strategies in current useor under evaluation in metastatic bone disease have been designed toprotect the bone matrix from increased bone degrading activity ofosteoclasts. An additional complication that presents in more than 80%of men with castration-resistant prostate cancer and metastasis bonedisease are osteosclerotic lesions—also known as bone-forming orosteoblastic lesions—or a combination of both, osteolytic andosteosclerotic lesions-also referred to as mixed lesions. Osteoscleroticlesions are typified by bone deposits with multiple layers of poorlyorganized type-I collagen fibrils that have a woven appearance andreduced mechanical strength.

Prostate cancer cells preserve, among each subtype,genome-aberration-induced transcriptional changes with high fidelity.The resulting dominant genes reveal molecular events that predict themetastatic outcome despite the existence of substantial genomic,transcriptional, translational, and biological heterogeneity in theoverall system. However, it is unknown whether the developmental historyof a cancer would result in different or common mediators ofsite-specific metastasis. Predisposing factors related to the cell oforigin may engender different rate-limiting barriers during metastasicprogression. Herein, we proposed the use of a new biomarker as aprognostic factor in primary tumors that predicts future bone metastasisevents. Moreover, we also propose the use of this gene as a potentialtherapeutic target to prevent, stop and cure prostate cancer derivedbone metastasis.

SUMMARY OF THE INVENTION

The present inventors have determined that identifying the balance ofsignals that affect disseminated prostate cancer cells bone metastasisprovides valuable information to establish the prognosis of, and forpreventive therapeutic intervention against, disease. Based on c-MAFexpression level and 16q22-24 bona fide ER+ breast cancer bonemetastasis genomic amplification, including MAF gene, contribution tobone metastasis, and particularly osteolytic bone metastasis, thepresent inventors identified that 16q22-24, including MAF gene, is alsoresponsible for driving the Prostate bone metastatic lesions, inparticular osteolytic Prostate bone metastasis.

The present inventors have identified—c-MAF as marker associated with agreater tendency of Prostate cancer to cause metastasis and,particularly, bone metastasis. This over-expression appears to be due toan amplification of the locus 16q22-q24 in which the c-MAF gene islocated.

The c-MAF expression levels were studied in a tissue microarray composedof Prostate primary tumor biopsies including 5 tumors that developmetastasis to the bone at any time, 3 that develop metastasis to othersites except bone and a minimum clinical follow up of 5 years and 29Prostate primary tumors that never develop metastasis with a minimumclinical follow up of 5 years, the c-MAF protein expression in tumorcells and biopsy correlates positively with different clinicalparameters, included metastasis and bone metastasis. Furthermore, theinventors have associated the amplification of the genomic locus16q22-q24, including the c-MAF gene, with the presence of metastasis insubjects with Prostate cancer and, in particular, in Prostate cancerthat form bone metastasis.

Thus, in a first aspect, the invention relates to an in vitro method forthe diagnosis of metastasis in a subject with Prostate cancer and/or theprognosis of the tendency to develop metastasis in a subject withProstate cancer which comprises

-   -   (i) quantifying the c-MAF gene or protein expression level or        copy number gain in a tumor sample of said subject and    -   (ii) comparing the expression level or copy number previously        obtained with the expression level or copies of said gene in a        control sample,        wherein if the expression levels of said gene are increased with        respect to the expression levels of said gene in the control        sample, then said subject has a positive diagnosis for        metastasis or a greater tendency to develop metastasis.

In a second aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with Prostate cancer whichcomprises

-   -   (i) quantifying the c-MAF gene or protein expression level in a        tumor sample of said subject and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression levels are increased with respect to        the expression levels of said gene in the control sample, then        said subject is susceptible to receive a therapy aiming to        prevent an/or treat the metastasis. In a particular aspect of        this method, the subject is then administered at least one        therapeutic drug that prevents, inhibits and/or treats the bone        metastasis, and        wherein if the expression level is not increased with respect to        said reference value, then said subject is not susceptible to        receive a therapy aiming to prevent, inhibit and/or treat the        bone metastasis. In a particular aspect of this method, the        subject is then not administered at least one therapeutic drug        that prevents, inhibits and/or treats the bone metastasis.

In a third aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with Prostate cancer withbone metastasis which comprises

-   -   (i) quantifying the c-MAF gene or protein expression level in a        bone metastatic tumor sample of said subject and    -   (ii) comparing the expression level obtained in step (i) with        the expression level of said gene in a control sample,        wherein if the c-MAF gene or protein expression levels are        increased with respect to the expression levels of said gene or        protein in the control sample, then said subject is susceptible        to receive a therapy aiming to prevent the bone degradation. In        a particular aspect of this method, the subject is then        administered at least one therapeutic drug that prevents,        inhibits and/or treats the bone metastasis, and        wherein if the c-MAF gene or protein expression level is not        increased with respect to said reference value, then said        subject is not susceptible to receive a therapy for preventing        the bone degradation. In a particular aspect of this method, the        subject is then not administered at least one therapeutic drug        that prevents, inhibits and/or treats the bone metastasis.

In a fourth aspect, the invention relates to an in vitro method for thediagnosis of metastasis in a subject with Prostate cancer and/or for theprognosis of the tendency to develop metastasis in a subject withProstate cancer which comprises determining if the c-MAF gene isamplified in a tumor tissue sample of said subject; wherein if said geneis amplified or translocated with respect to a control sample, then saidsubject has a positive diagnosis for metastasis or a greater tendency todevelop metastasis. In a particular aspect of this method, the subjectis then administered at least one therapeutic drug that prevents orinhibits the bone metastasis.

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering Prostate cancer,which comprises determining if the c-MAF gene is amplified in a sampleof said subject relative to a reference gene copy number wherein anamplification of the c-MAF gene with respect to said reference gene copynumber is indicative of a poor clinical outcome. In a particular aspectof this method, the subject is then administered at least onetherapeutic drug that prevents, inhibits and/or treats the bonemetastasis. If such amplification is not observed then the subject isnot administered at least one therapeutic drug that prevents, inhibitsand/or treats the bone metastasis. In another embodiment, the inventionrelates to an in vitro method for predicting the clinical outcome of apatient suffering prostate cancer which comprises determining if thec-MAF gene is translocated in a sample of said subject wherein atranslocation of the c-MAF gene (i.e. t(14,16)) is indicative of a poorclinical outcome.

In a fifth aspect, the invention relates to the use of a c-MAFinhibitory agent in the preparation of a medicinal product for treatingand/or preventing Prostate cancer metastasis, in particular bonemetastasis.

In another aspect, the invention relates to the use of an agent capableof avoiding or preventing bone degradation in the preparation of amedicinal product for the treatment of bone metastasis in a subjectsuffering Prostate cancer and having elevated c-MAF levels in ametastatic tumor tissue sample with respect to a control sample.

In another aspect, the invention relates to a kit for predicting bonemetastasis of a Prostate cancer in a subject suffering from said cancer,the kit comprising: a) means for determining translocation of the c-MAFgene in a sample of said subject; and b) means for comparing thetranslocation of c-MAF in said sample to a reference c-MAF sample. Theinvention also relates to the use of such kit to predict bone metastasisof a Prostate cancer in a subject suffering from said cancer. In oneembodiment, the subject is then administered or excluded at least onetherapeutic drug that prevents, inhibits and/or treats the bonemetastasis based on the results of using the kit.

In another aspect, the invention relates to a kit for predicting bonemetastasis of a Prostate cancer in a subject suffering from said cancer,the kit comprising: a) means for quantifying the amplification of c-MAFgene, 16q23 or 16q22-24 locus amplification or translocation in a sampleof said subject; and b) means for comparing the amplified level of c-MAFgene, 16q23 or 16q22-24 locus amplification or translocation in saidsample to a reference.

In another aspect, the invention relates to a kit for predicting theclinical outcome of a subject suffering from bone metastasis from aProstate cancer, the kit comprising: a) means for quantifying theexpression level of c-MAF in a sample of said subject; and b) means forcomparing the quantified expression level of c-MAF in said sample to areference c-MAF expression level. The invention also relates to the useof such kit to predict the clinical outcome of a subject suffering frombone metastasis from a Prostate cancer. In one embodiment, the subjectis then administered or excluded at least one therapeutic drug thatprevents, inhibits and/or treats the bone metastasis based on theresults of using the kit.

In another aspect, the invention relates to a kit for determining atherapy for a subject suffering from Prostate cancer, the kitcomprising: a) means for quantifying the expression level of c-MAF in asample of said subject; b) means for comparing the quantified expressionlevel of c-MAF in said sample to a reference c-MAF expression level; andc) means for determining a therapy for preventing and/or reducing bonemetastasis in said subject based on the comparison of the quantifiedexpression level to the reference expression level. The invention alsorelates to the use of such kit to determine a therapy for a subjectsuffering from Prostate cancer. In one embodiment, the subject is thenadministered or excluded at least one therapeutic drug that prevents,inhibits and/or treats the bone metastasis based on the results of usingthe kit.

In another aspect, the invention relates to a kit comprising: i) areagent for quantifying the expression level of c-MAF in a sample of asubject suffering from Prostate cancer, and ii) one or more c-MAF geneexpression level indices that have been predetermined to correlate withthe risk of bone metastasis. The invention also relates to the use ofsuch kit to predict bone metastasis of a prostate cancer in a subjectsuffering from said cancer. In one embodiment, the subject is thenadministered or excluded at least one therapeutic drug that prevents,inhibits and/or treats the bone metastasis based on the results of usingthe kit.

In another aspect, the invention relates to an in vitro method fortyping a sample of a subject suffering from Prostate cancer, the methodcomprising:

-   -   a) providing a sample from said subject;    -   b) quantifying the expression level of c-MAF in said sample;    -   c) typing said sample by comparing the quantified expression        level of c-MAF to a predetermined reference level of c-MAF        expression;        wherein said typing provides prognostic information related to        the risk of bone metastasis in said subject. In one embodiment,        the subject is administered or excluded at least one therapeutic        agent based on the prognostic information provided by the        typing.

In another aspect, the invention relates to a method for preventing orreducing the risk of bone metastasis in a subject suffering fromProstate cancer, said method comprising administering to said subject anagent that prevents or reduces bone metastasis, wherein said agent isadministered in accordance with a treatment regimen determined fromquantifying the expression level of c-MAF in said subject.

In another aspect, the invention relates to a method of classifying asubject suffering from Prostate cancer into a cohort, comprising: a)determining the expression level of c-MAF in a sample of said subject;b) comparing the expression level of c-MAF in said sample to apredetermined reference level of c-MAF expression; and c) classifyingsaid subject into a cohort based on said expression level of c-MAF inthe sample. In a particular aspect, the cohort is used for conducting aclinical trial.

DETAILED DESCRIPTION OF THE INVENTION Methods for the Diagnosis andPrognosis of Prostate Cancer Metastasis Based on c-MAF Expression Levels

The inventors have shown that the c-MAF gene and protein isoverexpressed in Prostate cancer metastasis, and that the c-MAFexpression levels in primary prostate tumors are correlated to differentclinical parameters of prostate cancer, particularly with recurrence andmetastasis probability. Thus, c-MAF overexpression is associated withthe onset and high risk of prostate tumor metastasis, particularly inbone. Therefore, c-MAF can be used as a marker for the diagnosis and/orprognosis of metastasis, in particular bone metastasis, in a subjectwith Prostate cancer.

Thus in one aspect, the invention relates to an in vitro method for thediagnosis of metastasis in a subject with Prostate cancer and/or for theprognosis of the tendency to develop metastasis in a subject withProstate cancer which comprises

-   -   (i) quantifying the c-MAF gene expression level in a tumor        sample (e.g., prostate tumor tissue, circulating prostate tumor        cell, circulating prostate tumor DNA) from said subject and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression level of said gene are increased with        respect to the expression level of said gene in the control        sample, then said subject has a positive diagnosis for        metastasis or a greater tendency to develop metastasis, in a        preferred site bone metastasis.

The c-MAF gene (v-maf musculoaponeurotic fibrosarcoma oncogene homologue(avian) also known as MAF or MGC71685) is a transcription factorcontaining a leucine zipper which acts like a homodimer or aheterodimer. Depending on the DNA binding site, the encoded protein canbe a transcriptional activator or repressor. The DNA sequence encodingc-MAF is described in the NCBI database under accession numberNG_(—)016440 (SEQ ID NO: 1) (coding)). The genomic sequence of c-MAF isset forth in SEQ ID NO:13. The methods of the present invention mayutilize either the coding sequence or the genomic DNA sequence. Twomessenger RNA are transcribed from said DNA sequence, each of the whichwill give rise to one of the two c-MAF protein isoforms, the α isoformand the β isoform. The complementary DNA sequences for each of saidisoforms are described, respectively, in the NCBI database underaccession numbers NM_(—)005360.4 (SEQ ID NO: 2) and NM_(—)001031804.2(SEQ ID NO: 3). Use of the c-MAF gene to predict the prognosis oftriple-negative and ER+ breast cancer is described in Int'l. Appl. No.PCT/IB2013/001204, which is incorporated herein by reference in itsentirety. Use of the c-MAF gene to predict the prognosis of thyroidcancer is described in U.S. Prov. Appl. No. 61/801,769, which isincorporated herein by reference in its entirety. Use of the c-MAF geneto predict the prognosis of renal cell carcinoma is described in U.S.Prov. Appl. No. 61/801,642, which is incorporated herein by reference inits entirety. The use of a gene of interest, including c-MAF and thec-MAF gene locus, to determine the prognosis of an individual sufferingbreast cancer is described in U.S. Prov. Appl. No. 61/801,718, which isincorporated herein by reference in its entirety. Use of the c-MAF geneto predict the prognosis of lung cancer is found in Int'l Appl. No.PCT/US2013/044584, which is incorporated herein by reference in itsentirety.

In the context of the present invention, “metastasis” is understood asthe propagation of a cancer from the organ where it started to adifferent organ. It generally occurs through the blood or lymphaticsystem. When the cancer cells spread and form a new tumor, the latter iscalled a secondary or metastatic tumor. The cancer cells forming thesecondary tumor are like those of the original tumor. If a Prostatecancer, for example, spreads (metastasizes) to the bone, the secondarytumor is formed of malignant Prostate cancer cells. The disease in thebone is metastatic Prostate cancer and not bone cancer. In a particularembodiment of the method of the invention, the metastasis is Prostatecancer which has spread (metastasized) to the bone.

In the present invention, “diagnosis of metastasis in a subject withProstate cancer” is understood as identifying a disease (metastasis) bymeans of studying its signs, i.e., in the context of the presentinvention by means of increased c-MAF gene expression levels (i.e.,overexpression) in the Prostate cancer tumor tissue with respect to acontrol sample.

In the present invention “prognosis of the tendency to developmetastasis in a subject with Prostate cancer” is understood as knowingbased on the signs if the Prostate cancer that said subject has willmetastasize in the future. In the context of the present invention, thesign is c-MAF gene overexpression in tumor tissue.

The method of the invention comprises in a first step quantifying thec-MAF gene expression level in a tumor tissue sample from a subject.

In a preferred embodiment, the first method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

As used herein, the term “subject” or “patient” refers to all animalsclassified as mammals and includes but is not limited to domestic andfarm animals, primates and humans, for example, human beings, non-humanprimates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.Preferably, the subject is a human man or woman of any age or race.

The terms “poor” or “good”, as used herein to refer to a clinicaloutcome, mean that the subject will show a favourable or unfavourableoutcome. As will be understood by those skilled in the art, such anassessment of the probability, although preferred to be, may not becorrect for 100% of the subjects to be diagnosed. The term, however,requires that a statistically significant portion of subjects can beidentified as having a predisposition for a given outcome. Whether aportion is statistically significant can be determined readily by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90% at least about 95%. The p-values are, preferably, 0.05, 0.01, 0.005,or 0.0001 or less. More preferably, at least about 60 percent, at leastabout 70 percent, at least about 80 percent or at least about 90 percentof the subjects of a population can be properly identified by the methodof the present invention.

In the present invention “tumor sample” is understood as a sample (e.g.,tumor tissue, circulating tumor cell, circulating tumor DNA) originatingfrom the primary Prostate cancer tumor. Said sample can be obtained byconventional methods, for example biopsy, using methods well known bythe persons skilled in related medical techniques. The methods forobtaining a biopsy sample include splitting a tumor into large pieces,or microdissection, or other cell separating methods known in the art.The tumor cells can additionally be obtained by means of cytologythrough aspiration with a small gauge needle. To simplify samplepreservation and handling, samples can be fixed in formalin and soakedin paraffin or first frozen and then soaked in a tissue freezing mediumsuch as OCT compound by means of immersion in a highly cryogenic mediumwhich allows rapid freezing.

As understood by the person skilled in the art, the gene expressionlevels can be quantified by measuring the messenger RNA levels of saidgene or of the protein encoded by said gene.

For this purpose, the biological sample can be treated to physically ormechanically break up the tissue or cell structure, releasing theintracellular components into an aqueous or organic solution forpreparing nucleic acids. The nucleic acids are extracted by means ofcommercially available methods known by the person skilled in the art(Sambroock, J., et al., “Molecular cloning: a Laboratory Manual”, 3rded., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3.)

Thus, the c-MAF gene expression level can be quantified from the RNAresulting from the transcription of said gene (messenger RNA or mRNA)or, alternatively, from the complementary DNA (cDNA) of said gene.Therefore, in a particular embodiment of the invention, thequantification of the c-MAF gene expression levels comprises thequantification of the messenger RNA of the c-MAF gene or a fragment ofsaid mRNA, complementary DNA of the c-MAF gene or a fragment of saidcDNA or the mixture thereof.

Virtually any conventional method can be used within the scope of theinvention for detecting and quantifying the mRNA levels encoded by thec-MAF gene or of the corresponding cDNA thereof. By way of non-limitingillustration, the mRNA levels encoded by said gene can be quantifiedusing conventional methods, for example, methods comprising mRNAamplification and the quantification of said mRNA amplification product,such as electrophoresis and staining, or alternatively, by Southern blotand using suitable probes, Northern blot and using specific probes ofthe mRNA of the gene of interest (c-MAF) or of the corresponding cDNAthereof, mapping with S1 nuclease, RT-PCR, hybridization, microarrays,etc., preferably by means of real time quantitative PCR using a suitablemarker. Likewise, the cDNA levels corresponding to said mRNA encoded bythe c-MAF gene can also be quantified by means of using conventionaltechniques; in this case, the method of the invention includes a stepfor synthesizing the corresponding cDNA by means of reversetranscription (RT) of the corresponding mRNA followed by theamplification and quantification of said cDNA amplification product.Conventional methods for quantifying expression levels can be found, forexample, in Sambrook et al., 2001. (cited ad supra). These methods areknown in the art and a person skilled in the art would be familiar withthe normalizations necessary for each technique. For example, theexpression measurements generated using multiplex PCR should benormalized by comparing the expression of the genes being measured to socalled “housekeeping” genes, the expression of which should be constantover all samples, thus providing a baseline expression to compareagainst or other control genes whose expression are known to bemodulated with cancer.

In a particular embodiment, the c-MAF gene expression levels arequantified by means of quantitative polymerase chain reaction (PCR) or aDNA, RNA array, or nucleotide hybridization technique.

In addition, the c-MAF gene expression level can also be quantified bymeans of quantifying the expression levels of the protein encoded bysaid gene, i.e., the c-MAF protein (c-MAF) [NCBI, accession numberO75444], or any functionally equivalent variant of the c-MAF protein.There are two c-MAF protein isoforms, the α isoform (NCBI,NP_(—)005351.2) made up of 403 amino acids (SEQ ID NO: 4) and the 8isoform (NP_(—)001026974.1) made up of 373 amino acids (SEQ ID NO: 5).The c-MAF gene expression level can be quantified by means ofquantifying the expression levels of any of the c-MAF protein isoforms.Thus, in a particular embodiment, the quantification of the levels ofthe protein encoded by the c-MAF gene comprises the quantification ofthe c-MAF protein.

In the context of the present invention, “functionally equivalentvariant of the c-MAF protein” is understood as (i) variants of the c-MAFprotein (SEQ ID NO: 4 or SEQ ID NO: 5) in which one or more of the aminoacid residues are substituted by a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue), wherein suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) variants comprising an insertion or a deletion ofone or more amino acids and having the same function as the c-MAFprotein, i.e., to act as a DNA binding transcription factor. Variants ofthe c-MAF protein can be identified using methods based on the capacityof c-MAF for promoting in vitro cell proliferation as shown ininternational patent application WO2005/046731 (hereby incorporated byreference in its entirety), based on the capacity of the so-calledinhibitor for blocking the transcription capacity of a reporter geneunder the control of cyclin D2 promoter or of a promoter containing thec-MAF responsive region (MARE or c-MAF responsive element) in cellsexpressing c-MAF as described in WO2008098351 (hereby incorporated byreference in its entirety), or based on the capacity of the so-calledinhibitor for blocking reporter gene expression under the control of theIL-4 promoter in response to the stimulation with PMA/ionomycin in cellsexpressing NFATc2 and c-MAF as described in US2009048117A (herebyincorporated by reference in its entirety).

The variants according to the invention preferably have sequencesimilarity with the amino acid sequence of any of the c-MAF proteinisoforms (SEQ ID NO: 4 or SEQ ID NO: 5) of at about least 50%, at leastabout 60%, at about least 70%, at least about 80%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at about least 98% or at about least 99%. The degree of similaritybetween the variants and the specific c-MAF protein sequences definedpreviously is determined using algorithms and computer processes whichare widely known by the persons skilled in the art. The similaritybetween two amino acid sequences is preferably determined using theBLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990)].

The c-MAF protein expression level can be quantified by any conventionalmethod which allows detecting and quantifying said protein in a samplefrom a subject. By way of non-limiting illustration, said protein levelscan be quantified, for example, by using antibodies with c-MAF bindingcapacity (or a fragment thereof containing an antigenic determinant) andthe subsequent quantification of the complexes formed. The antibodiesused in these assays may or may not be labeled. Illustrative examples ofmarkers that can be used include radioactive isotopes, enzymes,fluorophores, chemiluminescence reagents, enzyme substrates orcofactors, enzyme inhibitors, particles, dyes, etc. There is a widerange of known assays that can be used in the present invention whichuse unlabeled antibodies (primary antibody) and labeled antibodies(secondary antibody); these techniques include Western-blot or Westerntransfer, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), competitive EIA (competitive enzyme immunoassay),DAS-ELISA (double antibody sandwich ELISA), immunocytochemical andimmunohistochemical techniques, techniques based on the use of proteinmicroarrays or biochips including specific antibodies or assays based oncolloidal precipitation in formats such as dipsticks. Other ways fordetecting and quantifying said c-MAF protein include affinitychromatography techniques, ligand binding assays, etc. When animmunological method is used, any antibody or reagent that is known tobind to the c-MAF protein with a high affinity can be used for detectingthe amount thereof. This would include, but is not limited to, the useof an antibody, for example, polyclonal sera, supernatants of hybridomasor monoclonal antibodies, antibody fragments, Fv, Fab, Fab′ and F(ab′)2,scFv, humanized diabodies, triabodies, tetrabodies, antibodies,nanobodies, alphabodies, stapled peptides, and cyclopeptides. There arecommercial anti-c-MAF protein antibodies on the market which can be usedin the context of the present invention, such as for example antibodiesab427, ab55502, ab55502, ab72584, ab76817, ab77071 (Abcam plc, 330Science Park, Cambridge CB4 0FL, United Kingdom), the O75444 monoclonalantibody (Mouse Anti-Human MAF Azide free Monoclonal antibody,Unconjugated, Clone 6b8) of AbD Serotec, etc. There are many commercialcompanies offering anti-c-MAF antibodies, such as Abnova Corporation,Bethyl Laboratories, Bioworld Technology, GeneTex, etc.

In a particular embodiment, the c-MAF protein levels are quantifiedmeans of western blot, immunohistochemistry, ELISA or a protein array.

The first method of the invention comprises in a second step comparingthe c-MAF gene expression level obtained in the tumor sample (includingbut not limited to a primary tumor biopsy, circulating tumor cells andcirculating tumor DNA) from the subject with the expression level ofsaid gene in a control sample.

Once the c-MAF gene expression level in a tumor tissue sample, acirculating tumor cell or circulating tumor DNA from a subject withprostate cancer has been measured and compared with the control sample,if the expression level of said gene is increased with respect to itsexpression level in the control sample, then it can be concluded thatsaid subject has a positive diagnosis for metastasis or a greatertendency to develop metastasis.

The determination of the c-MAF gene expression level must be correlatedwith values of a control sample or reference sample. Depending on thetype of tumor to be analyzed, the exact nature of the control sample mayvary. Thus, in the event that a diagnosis is to be evaluated, then thereference sample is a tumor tissue sample from a subject with prostatecancer that has not metastasized or that corresponds to the median valueof the c-MAF gene expression levels measured in a tumor tissuecollection in biopsy samples from subjects with prostate cancer whichhave not metastasized.

Said reference sample is typically obtained by combining equal amountsof samples from a subject population. Generally, the typical referencesamples will be obtained from subjects who are clinically welldocumented and in whom the absence of metastasis is well characterized.In such samples, the normal concentrations (reference concentration) ofthe biomarker (c-MAF gene) can be determined, for example by providingthe mean concentration over the reference population. Variousconsiderations are taken into account when determining the referenceconcentration of the marker. Among such considerations are the age,weight, sex, general physical condition of the patient and the like. Forexample, equal amounts of a group of at least about 2, at least about10, at least about 100 to preferably more than 1000 subjects, preferablyclassified according to the foregoing considerations, for exampleaccording to various age categories, are taken as the reference group.The sample collection from which the reference level is derived willpreferably be formed by subjects suffering from the same type of canceras the patient object of the study (e.g., prostate cancer). Similarly,the reference value within a cohort of patients can be established usinga receiving operating curve (ROC) and measuring the area under the curvefor all de sensitivity and specificity pairs to determine which pairprovides the best values and what the corresponding reference value is.ROC is a standard statistical concept. A description can be found inStuart G. Baker “The Central Role of Receiver Operating Characteristic(ROC) curves in Evaluating Tests for the Early Detection of Cancer”Journal of The National Cancer Institute (2003) Vol 95, No. 7, 511-515.

Once this median or reference value has been established, the level ofthis marker expressed in tumor tissues from patients with this medianvalue can be compared and thus be assigned to the “increased” expressionlevel. Due to the variability among subjects (for example, aspectsreferring to age, race, etc.) it is very difficult (if not virtuallyimpossible) to establish absolute reference values of c-MAF expression.Thus, in particular embodiments the reference values for “increased” or“reduced” expression of the c-MAF expression are determined bycalculating the percentiles by conventional means which involvesperforming assays in one or several samples isolated from subjects whosedisease is well documented by any of the methods mentioned above thec-MAF expression levels. The “reduced” levels of c-MAF can thenpreferably be assigned to samples wherein the c-MAF expression levelsare equal to or lower than 50^(th) percentile in the normal populationincluding, for example, expression levels equal to or lower than the60^(th) percentile in the normal population, equal to or lower than the70^(th) percentile in the normal population, equal to or lower than the80^(th) percentile in the normal population, equal to or lower than the90^(th) percentile in the normal population, and equal to or lower thanthe 95^(th) percentile in the normal population. The “increased” c-MAFgene expression levels can then preferably be assigned to sampleswherein the c-MAF gene expression levels are equal to or greater thanthe 50^(th) percentile in the normal population including, for example,expression levels equal to or greater than the 60^(th) percentile in thenormal population, equal to or greater than the 70^(th) percentile inthe normal population, equal to or greater than the 80^(th) percentilein the normal population, equal to or greater than the 90^(th)percentile in the normal population, and equal to or greater than the95^(th) percentile in the normal population.

In the present invention “increased expression levels” or “increasedexpression level” is understood as the expression level when it refersto the levels of the c-MAF gene greater than those in a reference sampleor control sample. Particularly, a sample can be considered to have highc-MAF expression levels when the expression levels in the referencesample are at least about 1.1 times, 1.5 times, 5 times, 10 times, 20times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times, 100 times or even more with respect to the sample isolated fromthe patient.

In the context of the present invention, it is understood that “asubject has a positive diagnosis for metastasis” when the Prostatecancer suffered by said subject has metastasized to other organs of thebody, in a particular embodiment, to the bone.

In yet another embodiment, the metastasis to bone is an osteolytic bonemetastasis. As used herein, the expression “osteolytic bone metastasis”refers to a type of metastasis in which bone resorption (progressiveloss of the bone density) is produced in the proximity of the metastasisresulting from the stimulation of the osteoclast activity by the tumorcells and is characterized by severe pain, pathological fractures,hypercalcaemia, spinal cord compression and other syndromes resultingfrom nerve compression.

On the other hand, it is understood in the present invention that “asubject has a greater tendency to develop metastasis” when theprobabilities that the Prostate cancer suffered by the subject willmetastasize in the future are high.

The person skilled in the art will understand that the prediction of thetendency for a primary prostate tumor to metastasize is not intended tobe correct for all the subjects to be identified (i.e., for 100% of thesubjects). Nevertheless, the term requires enabling the identificationof a statistically significant part of the subjects (for example, acohort in a cohort study). Whether a part is statistically significantcan be determined in a simple manner by the person skilled in the artusing various well known statistical evaluation tools, for example, thedetermination of confidence intervals, determination of p values,Student's T test, Mann-Whitney test, etc. Details are provided in Dowdyand Wearden, Statistics for Research, John Wiley and Sons, New York1983. The preferred confidence intervals are at least about 90%, atleast about 95%, at least about 97%, at least 98% or at least 99%. The pvalues are preferably 0.1, 0.05, 0.01, 0.005 or 0.0001. More preferably,at least about 60%, at least about 70%, at least about 80% or at leastabout 90% of the subjects of a population can be suitably identified bythe method of the present invention.

As used herein, “agent for avoiding or preventing bone degradation”refers to any molecule capable of preventing, inhibiting, treating,reducing, or stopping bone degradation either by stimulating theosteoblast proliferation or inhibiting the osteoclast proliferation orfixing the bone structure.

As used herein, a “c-MAF inhibitory agent” refers to any moleculecapable of completely or partially inhibiting the c-MAF gene expression,both by preventing the expression product of said gene from beingproduced (interrupting the c-MAF gene transcription and/or blocking thetranslation of the mRNA coming from the c-MAF gene expression) and bydirectly inhibiting the c-MAF protein activity. C-MAF gene expressioninhibitors can be identified using methods based on the capacity of theso-called inhibitor to block the capacity of c-MAF to promote the invitro cell proliferation, such as shown in the international patentapplication WO2005/046731 (the entire contents of which are herebyincorporated by reference), based on the capacity of the so-calledinhibitor to block the transcription capacity of a reporter gene underthe control of the cyclin D2 promoter or of a promoter containing thec-MAF response region (MARE or c-MAF responsive element) in cells whichexpress c-MAF such as described in WO2008098351 (the entire contents ofwhich are hereby incorporated by reference) or based on the capacity ofthe so-called inhibitor to block the expression of a reporter gene underthe control of the IL-4 promoter in response to the stimulation withPMA/ionomycin in cells which express NFATc2 and c-MAF such as describedin US2009048117A (the entire contents of which is hereby incorporated byreference).

As used herein, Mammalian target of rapamycin (mTOR) or “mTor” refers tothose proteins that correspond to EC 2.7.11.1. mTor enzymes areserine/threonine protein kinases and regulate cell proliferation, cellmotility, cell growth, cell survival, and transcription.

As used herein, an “mTor inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the mTor gene expression, both bypreventing the expression product of said gene from being produced(interrupting the mTor gene transcription and/or blocking thetranslation of the mRNA coming from the mTor gene expression) and bydirectly inhibiting the mTor protein activity. Including inhibitors thathave a dual or more targets and among them mTor protein activity.

As used herein, “Src” refers to those proteins that correspond to EC2.7.10.2. Src is a non-receptor tyrosine kinase and a proto-oncogene.Src may play a role in cell growth and embryonic development.

As used herein, a “Src inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the Src gene expression, both bypreventing the expression product of said gene from being produced(interrupting the Src gene transcription and/or blocking the translationof the mRNA coming from the Src gene expression) and by directlyinhibiting the Src protein activity.

As used herein, “Prostaglandin-endoperoxide synthase 2”,“cyclooxygenase-2” or “COX-2” refers to those proteins that correspondto EC 1.14.99.1. COX-2 is responsible for converting arachidonic acid toprostaglandin endoperoxide H2.

As used herein, a “COX-2 inhibitor” refers to any molecule capable ofcompletely or partially inhibiting the COX-2 gene expression, both bypreventing the expression product of said gene from being produced(interrupting the COX-2 gene transcription and/or blocking thetranslation of the mRNA coming from the COX-2 gene expression) and bydirectly inhibiting the COX-2 protein activity.

As used herein “outcome” or “clinical outcome” refers to the resultingcourse of disease and/or disease progression and can be characterizedfor example by recurrence, period of time until recurrence, metastasis,period of time until metastasis, number of metastases, number of sitesof metastasis and/or death due to disease. For example a good clinicaloutcome includes cure, prevention of recurrence, prevention ofmetastasis and/or survival within a fixed period of time (withoutrecurrence), and a poor clinical outcome includes disease progression,metastasis and/or death within a fixed period of time.

“Predicting”, as used herein, refers to the determination of thelikelihood that the subject suffering lung cancer will developmetastasis to a distant organ. As used herein, “good prognosis”indicates that the subject is expected (e.g. predicted) to surviveand/or have no, or is at low risk of having, recurrence or distantmetastases within a set time period. The term “low” is a relative termand, in the context of this application, refers to the risk of the “low”expression group with respect to a clinical outcome (recurrence, distantmetastases, etc.). A “low” risk can be considered as a risk lower thanthe average risk for an heterogeneous cancer patient population. In thestudy of Paik et al. (2004), an overall “low” risk of recurrence wasconsidered to be lower than 15 percent. The risk will also vary infunction of the time period. The time period can be, for example, fiveyears, ten years, fifteen years or even twenty years after initialdiagnosis of cancer or after the prognosis was made.

As used herein, “poor prognosis” indicates that the subject is expectede.g. predicted to not survive and/or to have, or is at high risk ofhaving, recurrence or distant metastases within a set time period. Theterm “high” is a relative term and, in the context of this application,refers to the risk of the “high” expression group with respect to aclinical outcome (recurrence, distant metastases, etc.). A “high” riskcan be considered as a risk higher than the average risk for aheterogeneous cancer patient population. In the study of Paik et al.(2004), an overall “high” risk of recurrence was considered to be higherthan 15 percent. The risk will also vary in function of the time period.The time period can be, for example, five years, ten years, fifteenyears or even twenty years of initial diagnosis of cancer or after theprognosis was made.

“Reference value”, as used herein, refers to a laboratory value used asa reference for values/data obtained by laboratory examinations ofpatients or samples collected from patients. The reference value orreference level can be an absolute value; a relative value; a value thathas an upper and/or lower limit; a range of values; an average value; amedian value, a mean value, or a value as compared to a particularcontrol or baseline value. A reference value can be based on anindividual sample value, such as for example, a value obtained from asample from the subject being tested, but at an earlier point in time.The reference value can be based on a large number of samples, such asfrom a population of subjects of the chronological age matched group, orbased on a pool of samples including or excluding the sample to betested.

The term “treatment”, as used herein, refers to any type of therapy,which aims at terminating, preventing, ameliorating or reducing thesusceptibility to a clinical condition as described herein. In apreferred embodiment, the term treatment relates to prophylactictreatment (i.e. a therapy to reduce the susceptibility to a clinicalcondition), of a disorder or a condition as defined herein. Thus,“treatment,” “treating,” and their equivalent terms refer to obtaining adesired pharmacologic or physiologic effect, covering any treatment of apathological condition or disorder in a mammal, including a human. Theeffect may be prophylactic in terms of completely or partiallypreventing a disorder or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disorder and/or adverse effectattributable to the disorder. That is, “treatment” includes (1)preventing the disorder from occurring or recurring in a subject, (2)inhibiting the disorder, such as arresting its development, (3) stoppingor terminating the disorder or at least symptoms associated therewith,so that the host no longer suffers from the disorder or its symptoms,such as causing regression of the disorder or its symptoms, for example,by restoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating, orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain, or immunedeficiency.

As used herein, “sample” or “biological sample” means biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for determining the expression level of thec-MAF gene. The sample can be isolated from any suitable biologicaltissue or fluid such as, for example, tumor tissue, blood, blood plasma,serum, urine or cerebral spinal fluid (CSF).

As used herein, the term “expression level” of a gene as used hereinrefers to the measurable quantity of gene product produced by the genein a sample of the subject, wherein the gene product can be atranscriptional product or a translational product. Accordingly, theexpression level can pertain to a nucleic acid gene product such as mRNAor cDNA or a polypeptide gene product. The expression level is derivedfrom a subject's sample and/or a reference sample or samples, and canfor example be detected de novo or correspond to a previousdetermination. The expression level can be determined or measured, forexample, using microarray methods, PCR methods (such as qPCR), and/orantibody based methods, as is known to a person of skill in the art.

As used herein, the term “gene copy number” refers to the copy number ofa nucleic acid molecule in a cell. The gene copy number includes thegene copy number in the genomic (chromosomal) DNA of a cell. In a normalcell (non-tumoral cell), the gene copy number is normally two copies(one copy in each member of the chromosome pair). The gene copy numbersometimes includes half of the gene copy number taken from samples of acell population.

“Increased expression level” is understood as the expression level whenit refers to the levels of the c-MAF gene greater than those in areference sample or control sample. This increased levels can be causedwithout excluding other mechanisms by a gene or 16q23 or 16q22-24chromosomal locus amplification or translocation. Particularly, a samplecan be considered to have high c-MAF expression level when theexpression level in the sample isolated from the patient is at leastabout 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50times, 60 times, 70 times, 80 times, 90 times, 100 times or even morewith respect to the reference or control.

“Probe”, as used herein, refers to an oligonucleotide sequence that iscomplementary to a specific nucleic acid sequence of interest. In someembodiments, the probes may be specific to regions of chromosomes whichare known to undergo translocations. In some embodiments, the probeshave a specific label or tag. In some embodiments, the tag is afluorophore. In some embodiments, the probe is a DNA in situhybridization probe whose labeling is based on the stable coordinativebinding of platinum to nucleic acids and proteins. In some embodiments,the probe is described in U.S. patent application Ser. No. 12/067,532and U.S. patent application Ser. No. 12/181,399, which are incorporatedby reference in their entirety, or as described in Swennenhuis et al.“Construction of repeat-free fluorescence in situ hybridization probes”Nucleic Acids Research 40(3):e20 (2012).

“Tag” or “label”, as used herein, refers to any physical molecule whichis directly or indirectly associated with a probe, allowing the probe orthe location of the probed to be visualized, marked, or otherwisecaptured.

“Translocation”, as used herein, refers to the exchange of chromosomalmaterial in unequal or equal amounts between chromosomes. In some cases,the translocation is on the same chromosome. In some cases, thetranslocation is between different chromosomes. Translocations occur ata high frequency in many types of cancer, including breast cancer andleukemia. Translocations can be either primary reciprocal translocationsor the more complex secondary translocations. There are several primarytranslocations that involve the immunoglobin heavy chain (IgH) locusthat are believed to constitute the initiating event in many cancers.(Eychène, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer.2008. Nature Reviews: Cancer. 8: 683-693.)

“Polyploid” or “polyploidy”, as used herein, indicates that the cellcontains more than two copies of a gene of interest. In some instances,the gene of interest is MAF. In some embodiments, polyploidy isassociated with an accumulation of expression of the gene of interest.In some embodiments, polyploidy is associated with genomic instability.In some embodiments, the genomic instability may lead to chromosometranslocations.

“Whole genome sequencing”, as used herein, is a process by which theentire genome of an organism is sequenced at a single time. See, e.g.,Ng., P. C. amd Kirkness, E. F., Whole Genome Sequencing. 2010. Methodsin Molecular Biology. 628: 215-226.

“Exome sequencing”, as used herein, is a process by which the entirecoding region of the DNA of an organism is sequenced. In exomesequencing, the mRNA is sequenced. The untranslated regions of thegenome are not included in exome sequencing. See, e.g., Choi, M. et al.,Genetic diagnosis by whole exome capture and massively parallel DNAsequencing. 2009. PNAS. 106(45): 19096-19101.

“Tumor tissue sample” is understood as the tissue sample originatingfrom the prostate cancer tumor, including but not limited to circulatingtumor cells and circulating tumor DNA. Said sample can be obtained byconventional methods, for example biopsy, using methods well known bythe persons skilled in related medical techniques.

“Osteolytic bone metastasis” refers to a type of metastasis in whichbone resorption (progressive loss of the bone density) is produced inthe proximity of the metastasis resulting from the stimulation of theosteoclast activity by the tumor cells and is characterized by severepain, pathological fractures, hypercalcaemia, spinal cord compressionand other syndromes resulting from nerve compression.

Method for Designing Customized Therapy of the Invention in Patientswith Prostate Tumors

As is known in the state of the art, the treatment to be administered toa subject suffering from cancer depends on whether the latter is amalignant tumor, i.e., whether it has high probabilities of undergoingmetastasis, or whether the latter is a benign tumor. In the firstassumption, the treatment of choice is a systemic treatment such aschemotherapy and in the second assumption, the treatment of choice is alocalized treatment such as radiotherapy.

Therefore, as described in the present invention, given that the c-MAFgene overexpression in prostate cancer cells is related to the presenceof metastasis, the c-MAF gene expression levels allow making decisionsin terms of the most suitable therapy for the subject suffering saidcancer.

Thus, in another aspect the invention relates to an in vitro method fordesigning a customized therapy for a subject with prostate cancer, whichcomprises

-   -   (i) quantifying the c-MAF gene expression level in a tumor        sample of said subject and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression level are increased with respect to        the expression levels of said gene in the control sample, then        said subject is susceptible to receive a therapy aiming to        prevent and/or treat the metastasis. In a particular aspect of        this method, the subject is then administered at least one        therapeutic drug that prevents, inhibits and/or treats the bone        metastasis.        wherein if the c-MAF gene expression level is not increased with        respect to said reference value, then said subject is not        susceptible to receive a therapy for preventing the bone        degradation. In a particular aspect of this method, the subject        is then not administered at least one therapeutic drug that        prevents, inhibits and/or treats the bone metastasis.

In a particular embodiment, the metastasis is a bone metastasis. In amore preferred embodiment, the bone metastasis is osteolytic metastasis.

The terms and expressions “subject”, “prostate cancer”, “tumor sample”,“metastasis”, “determination of expression levels”, “c-MAF gene”,“increased expression levels” and “control sample” have been describedin detail in relation to the first method of the invention and areequally applicable to the second and third method of the invention.

The second method of the invention comprises in a first step quantifyingthe c-MAF gene expression level in a tumor sample in a subject sufferingfrom prostate cancer.

In a preferred embodiment, the second method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

In the case of the second method of the invention the sample is aprimary tumor tissue sample of the subject. In a second step, the c-MAFgene expression level obtained in the tumor sample of the subject iscompared with the expression level of said gene in a control sample. Thedetermination of the c-MAF gene expression levels must be related tovalues of a control sample or reference sample. Depending on the type oftumor to be analyzed, the exact nature of the control sample may vary.Thus preferably the reference sample is a tumor tissue sample of asubject with prostate cancer that has not metastasized or thatcorresponds to the median value of the c-MAF gene expression levelsmeasured in a tumor tissue collection in biopsy samples of subjects withprostate cancer which has not metastasized.

In yet another embodiment, an expression level of c-MAF which is abovethe average indicates increased risk of bone metastasis, the risk beingproportional to the levels of c-MAF expression, Thus, the risk of bonemetastasis in a subject suffering lung cancer is dose-dependent.

Once the c-MAF gene expression level in the sample have been measuredand compared with the control sample, if the expression level of saidgene are increased with respect to their expression levels in thecontrol sample, then it can be concluded that said subject issusceptible to receiving therapy aiming to prevent (if the subject hasyet to undergo metastasis) and/or treat metastasis (if the subject hasalready experienced metastasis). If such increased expression is notobserved then the subject is not administered at least one therapeuticdrug that prevents, inhibits and/or treats the bone metastasis.

As used herein, an “agent for avoiding or preventing bone degradation”refers to any molecule capable of treating or stopping bone degradationeither by stimulating the osteoblast proliferation or inhibiting theosteoclast proliferation. Illustrative examples of agents used foravoiding and/or preventing bone degradation include, although notlimited to:

-   -   Parathyroid hormone (PTH) and Parathyroid like hormone (PTHLH)        inhibitors (including blocking antibodies) or recombinant forms        thereof (teriparatide corresponding to the amino acids 7-34 of        PTH). This hormone acts by stimulating the osteoclasts and        increasing their activity.    -   Strontium ranelate: is an alternative oral treatment, and forms        part of the group of drugs called “dual action bone agents”        (DABAs) because they stimulate the osteoblast proliferation and        inhibit the osteoclast proliferation.    -   “Estrogen receptor modulators” (SERM) refers to compounds which        interfere or inhibit the binding of estrogens to the receptor,        regardless of the mechanism. Examples of estrogen receptor        modulators include, among others, estrogens progestagen,        estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424,        tamoxifen, idoxifene, L Y353381, LY117081, toremifene,        fluvestrant,        4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate        4,4′dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH646.    -   Calcitonin: directly inhibits the osteoclast activity through        the calcitonin receptor. The calcitonin receptors have been        identified on the surface of the osteoclasts.    -   Bisphosphonates: are a group of medicinal products used for the        prevention and the treatment of diseases with bone resorption        and reabsorption such as osteoporosis and cancer with bone        metastasis, the latter being with or without hypercalcaemia,        associated to breast cancer and prostate cancer. Examples of        bisphosphonates which can be used in the therapy designed by        means of the fifth method of the invention include, although not        limited to, nitrogenous bisphosphonates (such as pamidronate,        neridronate, olpadronate, alendronate, ibandronate, risedronate,        incadronate, zoledronate or zoledronic acid, etc.) and        non-nitrogenous bisphosphonates (such as etidronate, clodronate,        tiludronate, etc.).    -   “Cathepsin K inhibitors” refers to compounds which interfere in        the cathepsin K cysteine protease activity. Non-limiting        examples of cathepsin K inhibitors include        4-amino-pyrimidine-2-carbonitrile derivatives (described in the        International patent application WO 03/020278 under the name of        Novartis Pharma GMBH), pyrrolo-pyrimidines described in the        publication WO 03/020721 (Novartis Pharma GMBH) and the        publication WO 04/000843 (ASTRAZENECA AB) as well as the        inhibitors described in the publications PCT WO 00/55126 of Axys        Pharmaceuticals, WO 01/49288 of Merck Frosst Canada & Co. and        Axys Pharmaceuticals.    -   “DKK-1 (Dickkopf-1) inhibitor” as used herein refers to any        compound which is capable of reducing DKK-1 activity. DKK-1 is a        soluble Wnt pathway antagonist expressed predominantly in adult        bone and upregulated in myeloma patients with osteolytic        lesions. Agents targeting DKK-1 may play a role in preventing        osteolytic bone disease in multiple myeloma patients. BHQ880        from Novartis is a first-in-class, fully human, anti-DKK-1        neutralizing antibody. Preclinical studies support the        hypothesis that BHQ880 promotes bone formation and thereby        inhibits tumor-induced osteolytic disease (Ettenberg S. et al.,        American Association for Cancer Research Annual Meeting. Apr.        12-16, 2008; San Diego, Calif. Abstract).    -   “Dual MET and VEGFR2 inhibitor” as used herein refers to any        compound which is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. MET is        expressed not only in tumor cells and endothelial cells, but        also in osteoblasts (bone-forming cells) and osteoclasts        (bone-removing cells). HGF binds to MET on all of these cell        types, giving the MET pathway an important role in multiple        autocrine and paracrine loops. Activation of MET in tumor cells        appears to be important in the establishment of metastatic bone        lesions. At the same time, activation of the MET pathway in        osteoblasts and osteoclasts may lead to pathological features of        bone metastases, including abnormal bone growth (ie, blastic        lesions) or destruction (ie, lytic lesion. Thus, targeting the        MET pathway may be a viable strategy in preventing the        establishment and progression of metastatic bone lesions.        Cabozantinib (Exelixis, Inc), formerly known as XL184 (CAS        849217-68-1), is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. In multiple        preclinical studies cabozantinib has been shown to kill tumor        cells, reduce metastases, and inhibit angiogenesis (the        formation of new blood vessels necessary to support tumor        growth). Another suitable dual inhibitors are E7050        (N-[2-Fluoro-4-({2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonylaminopyridin-4-yl}oxy)phenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide        (2R,3R)-tartrate) (CAS 928037-13-2) or Foretinib (also known as        GSK1363089, XL880, CAS 849217-64-7).    -   “RANKL inhibitors” as used herein refer to any compound which is        capable of reducing the RANK activity. RANKL is found on the        surface of the osteoblast membrane of the stroma and        T-lymphocyte cells, and these T-lymphocyte cells are the only        ones which have demonstrated the capacity for secreting it. Its        main function is the activation of the osteoclasts, cells        involved in the bone resorption. The RANKL inhibitors can act by        blocking the binding of RANKL to its receptor (RANK), blocking        the RANK-mediated signaling or reducing the expression of RANKL        by blocking the transcription or the translation of RANKL. RANKL        antagonists or inhibitors suitable for use in the present        invention include, without limitation:        -   a suitable RANK protein which is capable of binding RANKL            and which comprises the entire or a fragment of the            extracellular domain of a RANK protein. The soluble RANK may            comprise the signal peptide and the extracellular domain of            the murine or human RANK polypeptides, or alternatively, the            mature form of the protein with the signal peptide removed            can be used.        -   Osteoprotegerin or a variant thereof with RANKL-binding            capacity.        -   RANKL-specific antisense molecules        -   Ribozymes capable of processing the transcribed products of            RANKL        -   Specific anti-RANKL antibodies. “Anti-RANKL antibody or            antibody directed against RANKL” is understood herein as all            that antibody which is capable of binding specifically to            the ligand of the activating receptor for the nuclear factor            KB (RANKL) inhibiting one or more RANKL functions. The            antibodies can be prepared using any of the methods which            are known by the person skilled in the art. Thus, the            polyclonal antibodies are prepared by means of immunizing an            animal with the protein to be inhibited. The monoclonal            antibodies are prepared using the method described by            Kohler, Milstein et al. (Nature, 1975, 256: 495). Antibodies            suitable in the context of the present invention include            intact antibodies which comprise a variable antigen binding            region and a constant region, fragments “Fab”, “F(ab′)2” and            “Fab′”, Fv, scFv, diabodies and bispecific antibodies.        -   Specific anti-RANKL nanobodies. Nanobodies are            antibody-derived therapeutic proteins that contain the            unique structural and functional properties of            naturally-occurring heavy-chain antibodies. The Nanobody            technology was originally developed following the discovery            that camelidae (camels and llamas) possess fully functional            antibodies that lack light chains. The general structure of            nanobodies is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        -   wherein FR1 to FR4 are the framework regions 1 to 4 CDR1 to            CDR3 are the complementarity determining regions 1 to 3.            These heavy-chain antibodies contain a single variable            domain (VHH) and two constant domains (CH2 and CH3).            Importantly, the cloned and isolated VHH domain is a            perfectly stable polypeptide harbouring the full            antigen-binding capacity of the original heavy-chain            antibody. These newly discovered VHH domains with their            unique structural and functional properties form the basis            of a new generation of therapeutic antibodies which Ablynx            has named Nanobodies.

In one embodiment, the RANKL inhibitor is selected from the groupconsisting of a RANKL specific antibody, a RANKL specific nanobody andosteoprotegerin. In a specific embodiment, the anti-RANKL antibody is amonoclonal antibody. In a yet more specific embodiment, the anti-RANKLantibody is Denosumab (Pageau, Steven C. (2009). mAbs 1 (3): 210-215,CAS number 615258-40-7) (the entire contents of which are herebyincorporated by reference). Denosumab is a fully human monoclonalantibody which binds to RANKL and prevents its activation (it does notbind to the RANK receptor). Various aspects of Denosumab are covered byU.S. Pat. Nos. 6,740,522; 7,411,050; 7,097,834; 7,364,736 (the entirecontents of each of which are hereby incorporated by reference in theirentirety). In another embodiment, the RANKL inhibitor an antibody,antibody fragment, or fusion construct that binds the same epitope asDenosumab.

In a preferred embodiment, the anti-RANKL nanobody is any of thenanobodies as described in WO2008142164, (the contents of which areincorporated in the present application by reference). In a still morepreferred embodiment, the anti-RANKL antibody is the ALX-0141 (Ablynx).ALX-0141 has been designed to inhibit bone loss associated withpost-menopausal osteoporosis, reumatoid arthritis, cancer and certainmedications, and to restore the balance of healthy bone metabolism.

In a preferred embodiment, the agent preventing the bone degradation isselected from the group consisting of a bisphosphonate, a RANKLinhibitor, PTH and PTHLH inhibitor or a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, Radium-223, calcitonin, and a cathepsin K inhibitor. In amore preferred embodiment the agent preventing the bone degradation is abisphosphonate. In a yet more preferred embodiment, the bisphosphonateis the zoledronic acid.

In one embodiment, a CCR5 antagonist is administered to prevent orinhibit metastasis of the primary prostate cancer tumor to bone. In oneembodiment, the CCR5 antagonist is a large molecule. In anotherembodiment, the CCR5 antagonist is a small molecule. In someembodiments, the CCR5 antagonist is Maraviroc. In some embodiments, theCCR5 antagonist is Vicriviroc. In some aspects, the CCR5 antagonist isAplaviroc. In some aspects, the CCR5 antagonist is a spiropiperidineCCR5 antagonist. (Rotstein D. M. et al. 2009. Spiropiperidine CCR5antagonists. Bioorganic & Medicinal Chemistry Letters. 19 (18):5401-5406. In some embodiments, the CCR5 antagonist is INCB009471(Kuritzkes, D. R. 2009. HIV-1 entry inhibitors: an overview. Curr. Opin.HIV AIDS. 4(2): 82-7).

In a preferred embodiment the dual MET and VEGFR2 inhibitor is selectedfrom the group consisting of Cabozantinib, Foretinib and E7050.

In another aspect, the treatment is an mTor inhibitor. In some aspects,the mTor inhibitor is a dual mTor/PI3kinase inhibitor. In some aspects,the mTor inhibitor is used to prevent or inhibit metastasis. In someaspects the mTor inhibitor is selected from the group consisting of:ABI009 (sirolimus), rapamycin (sirolimus), Abraxane (paclitaxel), Absorb(everolimus), Afinitor (everolimus), Afinitor with Gleevec, AS703026(pimasertib), Axxess (umirolimus), AZD2014, BEZ235, Biofreedom(umirolimus), BioMatrix (umirolimus), BioMatrix flex (umirolimus),CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUSNEICH(sirolimus), Curaxin CBLC102 (mepacrine), DE109 (sirolimus), DS3078,Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus), Femara(letrozole), Hocena (antroquinonol), INK128, Inspiron (sirolimus),IPI504 (retaspimycin hydrochloride), KRN951 (tivozanib), ME344, MGA031(teplizumab), MiStent SES (sirolimus), MKCl, Nobori (umirolimus),OSI027, OVI123 (cordycepin), Palomid 529, PF04691502, Promus Element(everolimus), PWT33597, Rapamune (sirolimus), Resolute DES(zotarolimus), RG7422, SAR245409, SF1126, SGN75 (vorsetuzumabmafodotin), Synergy (everolimus), Taltorvic (ridaforolimus), Tarceva(erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V(everolimus), Zomaxx (zotarolimus), Zortress (everolimus), ZotarolimusEluting Peripheral Stent MEDTRONIC (zotarolimus), AP23841, AP24170,ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371,EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602,Sirolimus PNP SAMYANG BIOPHARMACEUTICALS (sirolimus), TOP216, VLI27,VS5584, WYE125132, XL388, Advacan (everolimus), AZD8055, Cypher SelectPlus Sirolimus eluting Coronary Stent (sirolimus), Cypher Sirolimuseluting coronary stent (sirolimus), Drug Coated Balloon (sirolimus),E-Magic Plus (sirolimus), Emtor (sirolimus), Esprit (everolimus),Evertor (everolimus), HBF0079, LCP-Siro (sirolimus), Limus CLARIS(sirolimus), mTOR Inhibitor CELLZOME, Nevo Sirolimus eluting CoronaryStent (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus),ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus),Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (sirolimus),Sirolimus WATSON (sirolimus) Siropan (sirolimus), Sirova (sirolimus),Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON(tacrolimus), TAFA93, Temsirolimus ACCORD (temsirolimus), TemsirolimusSANDOZ (temsirolimus), TOP216, Xience Prime (everolimus), Xience V(everolimus). In a specific aspect the mTor inhibitor is Afinitor(everolimus)(http://www.afinitor.com/index.jsp?usertrack.filter_applied=true&Novald=4029462064338207963;last accessed Nov. 28, 2012). In another aspect, mTor inhibitors can beidentified through methods known in the art. (See, e.g., Zhou, H. et al.Updates of mTor inhibitors. 2010. Anticancer Agents Med. Chem. 10(7):571-81, which is herein incorporated by reference). In some aspects, themTor inhibitor is used to treat or prevent or inhibit metastasis in apatient with advanced prostate cancer. In some aspects, the mTorinhibitor is used in combination with a second treatment. In someaspects, the second treatment is any treatment described herein.

In another aspect, the treatment is a Src kinase inhibitor. In someaspects, the Src inhibitor is used to prevent or inhibit metastasis. Insome aspects, the Src kinase inhibitor is selected from the group:AZD0530 (saracatinib), Bosulif (bosutinib), ENMD981693, KD020, KX01,Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588,AZD0424, c-Src Kinase Inhibitor KISSE1, CU201, KX2361, SKS927, SRN004,SUNK706, TG100435, TG100948, AP23451, Dasatinib HETERO (dasatinib),Dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src Kinase InhibitorKINEX, VX680, (tozasertib lactate), XL228, and SUNK706. In someembodiments, the Src kinase inhibitor is dasatinib. In another aspect,Src kinase inhibitors can be identified through methods known in the art(See, e.g., Sen, B. and Johnson, F. M. Regulation of Src Family Kinasesin Human Cancers. 2011. J. Signal Transduction. 2011: 14 pages, which isherein incorporated by reference). In some aspects, the Src kinaseinhibitor is used to treat or prevent or inhibit metastasis in a patientthat is positive for the SRC-responsive signature (SRS). In someaspects, the Src kinase inhibitor is used to treat or prevent or inhibitmetastasis in a patient with advanced prostate cancer. In some aspects,the Src kinase inhibitor is used in combination with a second treatment.In some aspects, the second treatment is any treatment described herein.

In another aspect, the treatment is a COX-2 inhibitor. In some aspects,the COX-2 inhibitor is used to prevent or inhibit metastasis. In someaspects, the COX-2 inhibitor is selected from the group: ABT963,Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketorolactromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine,naproxen sodium), BAY11902 (piroxicam), BCIBUCH001 (ibuprofen),Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD(diclofenac), Diractin (ketoprofen), GW406381, HCT1026(nitroflurbiprofen), Hyanalgese-D (diclofenac), HydrocoDex(acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER(ibuprofen sodium), Ibuprofen with Acetaminophen PFIZER (acetaminophen,ibuprofen), Impracor (ketoprofen), IP880 (diclofenac), IP940(indomethacin), ISV205 (diclofenac sodium), JNS013 (acetaminophen,tramadol hydrochloride), Ketoprofen TDS (ketoprofen), LTNS001 (naproxenetemesil), Mesalamine SALIX (mesalamine), Mesalamine SOFAR (mesalamine),Mesalazine (mesalamine), ML3000 (licofelone), MRX7EAT (etodolac),Naproxen IROKO (naproxen), NCX4016 (nitroaspirin), NCX701(nitroacetaminophen), Nuprin SCOLR (ibuprofen), OMS103HP (amitriptylinehydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease(diclofenac), OxycoDex (dextromethorphan, oxycodone), P54, PercoDex(acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen,phosphatidyl choline), PSD508, R-Ketoprofen (ketoprofen), Remura(bromfenac sodium), ROX828 (ketorolac tromethamine), RP19583 (ketoprofenlysine), RQ00317076, SDX101 (R-etodolac), TDS943 (diclofenac sodium),TDT070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063(S-flurbiprofen), UR8880 (cimicoxib), V0498TA01A (ibuprofen), VT122(etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene),XP21B (diclofenac potassium), XP21L (diclofenac potassium), Zoenasa(acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed-P, Acular,Acular LS, Acular PF, Acular X, Acuvail, Advil, Advil Allergy Sinus,Advil Cold and Sinus, Advil Congestion Relief, Advil PM, Advil PMCapsule, Air Salonpas, Airtal, Alcohol-Free NyQuil Cold & Flu Relief,Aleve, Aleve ABDI IBRAHIM, Aleve-D, Alka-Seltzer, Alka-Seltzer BAYER,Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-SeltzerOriginal, Alka-Seltzer Plus, Alka-Seltzer plus Cold and Cough,Alka-Seltzer plus Cold and Cough Formula, Alka-Seltzer Plus Day andNight Cold Formula, Alka-Seltzer Plus Day Non-Drowsy Cold Formula,Alka-Seltzer Plus Flu Formula, Alka-Seltzer Plus Night Cold Formula,Alka-Seltzer Plus Sinus Formula, Alka-Seltzer Plus Sparkling OriginalCold Formula, Alka-Seltzer PM, Alka-Seltzer Wake-Up Call, Anacin,Anaprox, Anaprox MINERVA, Ansaid, Apitoxin, Apranax, Apranax abdi,Arcoxia, Arthritis Formula Bengay, Arthrotec, Asacol, Asacol HD, AsacolMEDUNA ARZNEIMITTEL, Asacol ORIFARM, Aspirin BAYER, Aspirin Complex,Aspirin Migran, AZD3582, Azulfidine, Baralgan M, BAY1019036, BAY987111,BAY11902, BCIBUCH001, Benadryl Allergy, Benadryl Day and Night, Benzylin4 Flu, Benzylin Cold and Flu, Benzylin Cold and Flu Day and Night,Benzylin Cold and Sinus Day and Night, Benzylin Cold and Sinus Plus,Benzylin Day and Night Cold and Flu Relief, Benzylinl All-In-One,Brexin, Brexin ANGELINI, Bromday, Bufferin, Buscopan Plus, Caldolor,Calmatel, Cambia, Canasa, Capoxigem, Cataflam, Celebrex, CelebrexORIFARM, Children's Advil Allergy Sinus, Children's Tylenol, Children'sTylenol Cough and Runny Nose, Children's Tylenol plus cold, Children'sTylenol plus Cold and Cough, Children's Tylenol plus cold and stuffynose, Children's Tylenol plus Flu, Children's Tylenol plus cold &allergy, Children's Tylenol plus Cough & Runny Nose, Children's Tylenolplus Cough & Sore Throat, Children's Tylenol plus multi symptom cold,Clinoril, Codral Cold and Flu, Codral Day and Night Day Tablets, CodralDay and Night Night Tablets, Codral Nightime, Colazal, Combunox, ContacCold plus Flu, Contac Cold plus Flu Non-Drowsy, Coricidin D, CoricidinHBP Cold and Flu, Coricidin HBP Day and Night Multi-Symptom Cold,Coricidin HBP Maximum Strength Flu, Coricidin HBP NighttimeMulti-Symptom Cold, Coricidin II Extra Strength Cold and Flu, CS502,CS670, Daypro, Daypro Alta, DDSO6C, Demazin Cold and Flu, Demazin Cough,Cold and Flu, Demazin day/night Cold and Flu, Demazin PE Cold and Flu,Demazin PE day/night Cold and Flu, Diclofenac HPBCD, Dimetapp DayRelief, Dimetapp Multi-Symptom Cold and Flu, Dimetapp Night Relief,Dimetapp Pain and Fever Relief, Dimetapp PE Sinus Pain, Dimetapp PESinus Pain plus Allergy, Dipentum, Diractin, Disprin Cold ‘n’ Fever,Disprin Extra, Disprin Forte. Disprin Plus, Dristan Cold, DristanJunior, Drixoral Plus, Duexis, Dynastat, Efferalgan, Efferalgan PlusVitamin C, Efferalgan Vitamin C, Elixsure IB, Excedrin Back and Body,Excedrin Migraine, Excedrin PM, Excedrin Sinus Headache, ExcedrinTension Headache, Falcol, Fansamac, Feldene, FeverAll, Fiorinal,Fiorinal with Codeine, Flanax, Flector Patch, Flucam, Fortagesic,Gerbin, Giazo, Gladio, Goody's Back and Body Pain, Goody's Cool Orange,Goody's Extra Strength, Goody's PM, Greaseless Bengay, GW406381,HCT1026, He Xing Yi, Hyanalgese-D, HydrocoDex, Ibuprofen Sodium PFIZER,Ibuprofen with, Acetaminophen PFIZER, Icy Hot SANOFI AVENTIS, Impracor,Indocin, Indomethacin APP PHARMA, Indomethacin MYLAN, Infants' Tylenol,IP880, IP940, Iremod, ISV205, JNS013, Jr. Tylenol, Junifen, JuniorStrength Advil, Junior Strength Motrin, Ketoprofen TDS, Lemsip Max,Lemsip Max All in One, Lemsip Max All Night, Lemsip Max Cold and Flu,Lialda, Listerine Mouth Wash, Lloyds Cream, Lodine, Lorfit P, Loxonin,LTNS001, Mersyndol, Mesalamine SALIX, Mesalamine SOFAR, Mesalazine,Mesasal GLAXO, Mesasal SANOFI, Mesulid, Metsal Heat Rub, Midol Complete,Midol Extended Relief, Midol Liquid Gels, Midol PM, Midol Teen Formula,Migranin COATED TABLETS, ML3000, Mobic, Mohrus, Motrin, Motrin Cold andSinus Pain, Motrin PM, Movalis ASPEN, MRX7EAT, Nalfon, Nalfon PEDINOL,Naprelan, Naprosyn, Naprosyn RPG LIFE SCIENCE, Naproxen IROKO, NCX4016,NCX701, NeoProfen LUNDBECK, Nevanac, Nexcede, Niflan, Norgesic MEDICIS,Novalgin, Nuprin SCOLR, Nurofen, Nurofen Cold and Flu, Nurofen MaxStrength Migraine, Nurofen Plus, Nuromol, NyQuil with Vitamin C, Ocufen,OMS103HP, Oralease, Orudis ABBOTT JAPAN, Oruvail, Osteluc, OxycoDex,P54, Panadol, Panadol Actifast, Paradine, Paramax, Parfenac, Pedea,Pennsaid, Pentasa, Pentasa ORIFARM, Peon, Percodan, Percodan-Demi,PercoDex, Percogesic, Perfalgan, PL2200, PL3100, Ponstel, Prexige,Prolensa, PSD508, R-Ketoprofen, Rantudil, Relafen, Remura, Robaxisal,Rotec, Rowasa, ROX828, RP19583, RQ00317076, Rubor, Salofalk, Salonpas,Saridon, SDX101, Seltouch, sfRowasa, Shinbaro, Sinumax, Sinutab,Sinutab, sinus, Spalt, Sprix, Strefen, Sudafed Cold and Cough, SudafedHead Cold and Sinus, Sudafed PE Cold plus Cough, Sudafed PE Pressureplus Pain, Sudafed PE, Severe Cold, Sudafed PE Sinus Day plus NightRelief Day Tablets, Sudafed PE Sinus Day plus Night Relief NightTablets, Sudafed PE Sinus plus Anti-inflammatory Pain Relief, SudafedSinus Advance, Surgam, Synalgos-DC, Synflex, Tavistallergy/sinus/headache, TDS943, TDT070, Theraflu Cold and Sore Throat,Theraflu Daytime Severe Cold and Cough, Theraflu Daytime Warming Relief,Theraflu Warming Relief Caplets Daytime Multi-Symptom Cold, TherafluWarming Relief Cold and Chest Congestion, Thomapyrin, Thomapyrin C,Thomapyrin Effervescent, Thomapyrin Medium, Tilcotil, Tispol, Tolectin,Toradol, TPR100, TQ1011, Trauma-Salbe, Trauma-Salbe Kwizda, Treo,Treximet, Trovex, TT063, Tylenol, Tylenol Allergy Multi-Symptom, TylenolBack Pain, Tylenol Cold & Cough Daytime, Tylenol Cold & Cough Nighttime,Tylenol Cold and Sinus Daytime, Tylenol Cold and Sinus Nighttime,Tylenol Cold Head Congestion Severe, Tylenol Cold Multi Symptom Daytime,Tylenol Cold Multi Symptom Nighttime Liquid, Tylenol Cold Multi SymptomSevere, Tylenol Cold Non-Drowsiness Formula, Tylenol Cold SevereCongestion Daytime, Tylenol Complete Cold, Cough and Flu Night time,Tylenol Flu Nighttime, Tylenol Menstrual, Tylenol PM, Tylenol SinusCongestion & Pain Daytime, Tylenol Sinus Congestion & Pain Nighttime,Tylenol Sinus Congestion & Pain Severe, Tylenol Sinus Severe CongestionDaytime, Tylenol Ultra Relief, Tylenol with Caffeine and Codeinephosphate, Tylenol with Codeine phosphate, Ultra Strength Bengay Cream,Ultracet, UR8880, V0498TA01A, Vicks NyQuil Cold and Flu Relief,Vicoprofen, Vimovo, Voltaren Emulgel, Voltaren GEL, Voltaren NOVARTISCONSUMER HEALTH GMBH, Voltaren XR, VT122, Xefo, Xefo Rapid, Xefocam,Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, Zipsor, and Zoenasa. In anotheraspect, COX-2 inhibitors can be identified through methods known in theart (See, e.g., Dannhardt, G. and Kiefer, W. Cyclooxygenaseinhibitors-current status and future prospects. 2001. Eur. J. Med. Chem.36: 109-126, which is herein incorporated by reference). In someaspects, the COX-2 inhibitor is used to treat or prevent or inhibitmetastasis in a patient with advanced prostate cancer. In some aspects,the COX-2 inhibitor is used in combination with a second treatment. Insome aspects, the second treatment is any treatment described herein. Insome aspects, the COX-2 inhibitor is used in combination with a secondtreatment selected from the group consisting of: Denosumab, Zometa(http://www.us.zometa.com/index.jsp?usertrack.filter_applied=true&Novald=2935376934467633633; last accessed Dec. 2, 2012),Carbozantinib or Cabozantinib, Antibody or peptide blocking PTHLH(parathyroid hormone like hormone) or PTHrP (parathyroid hormone relatedprotein).

In one embodiment, the treatment is Radium 223. In a preferredembodiment the Radium 223 therapy is Alpharadin (aka, Xofigo)(radium-223 dichloride). Alpharadin uses alpha radiation from radium-223decay to kill cancer cells. Radium-223 naturally self-targets to bonemetastases by virtue of its properties as a calcium-mimic. Alpharadiation has a very short range of 2-10 cells (when compared to currentradiation therapy which is based on beta or gamma radiation), andtherefore causes less damage to surrounding healthy tissues(particularly bone marrow). With similar properties to calcium,radium-223 is drawn to places where calcium is used to build bone in thebody, including the site of faster, abnormal bone growth—such as thatseen in the skeletal metastases of men with advanced,castration-resistant prostate cancer. Radium-223, after injection, iscarried in the bloodstream to sites of abnormal bone growth. The placewhere a cancer starts in the body is known as the primary tumor. Some ofthese cells may break away and be carried in the bloodstream to anotherpart of the body. The cancer cells may then settle in that part of thebody and form a new tumor. If this happens it is called a secondarycancer or a metastasis. Most patients with late stage prostate cancersuffer the maximum burden of disease in their bones. The aim withradium-223 is to selectively target this secondary cancer. Anyradium-223 not taken-up in the bones is quickly routed to the gut andexcreted.

Alternatively a combined treatment can be carried out in which more thanone agent from those mentioned above are combined to treat and/orprevent the metastasis or said agents can be combined with othersupplements, such as calcium or vitamin D or with a hormone treatment.

When the cancer has metastasized, systemic treatments including but notlimited to chemotherapy, hormone treatment, immunotherapy, or acombination thereof are used. Additionally, radiotherapy and/or surgerycan be used. The choice of treatment generally depends on the type ofprimary cancer, the size, the location of the metastasis, the age, thegeneral health of the patient and the types of treatments usedpreviously.

The systemic treatments are those that reach the entire body:

-   -   Chemotherapy is the use of medicaments to destroy cancer cells.        The medicaments are generally administered through oral or        intravenous route. Sometimes, chemotherapy is used together with        radiation treatment.    -   Hormone therapy is based on the fact that some hormones promote        cancer growth. For example, estrogen in women produced by the        ovaries sometimes promotes the breast cancer growth. There are        several ways for stopping the production of these hormones. A        way is to remove the organs producing them: the ovaries in the        case of women, the testicles in the case of the men. More        frequently, medicaments to prevent these organs from producing        the hormones or to prevent the hormones from acting on the        cancer cells can be used.    -   Immunotherapy is a treatment that aids the immune system itself        of the patient to combat cancer. There are several types of        immunotherapy which are used to treat metastasis patients. These        include but are not limited to cytokines, monoclonal antibodies        and antitumor vaccines.        Method for Designing Customized Therapy of the Invention in        Prostate Cancer Patients with Bone Metastasis

Patients suffering prostate cancer which has already metastasized to thebone and in which there are elevated c-MAF levels may particularlybenefit from therapies aimed at preventing the bone degradation causedby the increased osteoclastic activity.

Thus, in another aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject with prostate cancer withbone metastasis which comprises

-   -   (i) quantifying the c-MAF gene expression level in a metastatic        tumor sample from bone of said subject, and    -   (ii) comparing the expression level previously obtained with the        expression level of said gene in a control sample,        wherein if the expression levels are increased with respect to        the expression levels of said gene in the control sample, then        said subject is susceptible to receive a therapy aiming to        prevent the bone degradation.        wherein if the expression level is not increased with respect to        said reference value, then said subject is not susceptible to        receive a therapy aiming to prevent and/or treat the bone        metastasis.

The terms and expressions “subject”, “prostate cancer”, “tumor sample”,“metastasis”, “determination of expression levels”, “c-MAF gene”,“increased expression levels” and “control sample” have been describedin detail in relation to the first method of the invention and areequally applicable to the second and third method of the invention.

In a preferred embodiment, the bone metastasis is osteolytic metastasis.

The third method of the invention comprises in a first step, quantifyingthe c-MAF gene expression level in a tumor sample in a subject sufferingprostate cancer. In the case of the third method of the invention, thesample is a tissue sample from bone metastasis.

In a preferred embodiment, the third method of the invention comprisesquantifying only the c-MAF gene expression level as a single marker,i.e., the method does not involve determining the expression level ofany additional marker.

In a second step the c-MAF gene expression level obtained in the tumorsample of the subject is compared with the expression level of said genein a control sample. The determination of the c-MAF gene expressionlevels must be correlated to values of a control sample or referencesample. Depending on the type of tumor to be analyzed, the exact natureof the control sample may vary. Thus, in the case involving the thirdmethod of the invention, then the reference sample is a tumor tissuesample of subject with prostate cancer who has not suffered metastasisor that correspond to the median value of the c-MAF gene expressionlevel measured in a tumor tissue collection in biopsy samples ofsubjects with prostate cancer who has not suffered metastasis.

Once the c-MAF gene expression level in the sample is measured andcompared with the control sample, if the expression level of said geneare increased with respect to its expression level in the controlsample, then it can be concluded that said subject is susceptible toreceive a therapy aiming to avoid or prevent bone degradation.

As used herein, an “agent for avoiding or preventing bone degradation”refers to any molecule capable of treating or stopping bone degradationeither by stimulating the osteoblast proliferation or inhibiting theosteoclast proliferation. Illustrative examples of agents used foravoiding and/or preventing bone degradation include, although notlimited to:

-   -   Parathyroid hormone (PTH) and Parathyroid like hormone (PTHLH)        inhibitors (including blocking antibodies) or recombinant forms        thereof (teriparatide corresponding to the amino acids 7-34 of        PTH). This hormone acts by stimulating the osteoclasts and        increasing their activity.    -   Strontium ranelate: is an alternative oral treatment, and forms        part of the group of drugs called “dual action bone agents”        (DABAs) because they stimulate the osteoblast proliferation and        inhibit the osteoclast proliferation.    -   “Estrogen receptor modulators” (SERM) refers to compounds which        interfere or inhibit the binding of estrogens to the receptor,        regardless of the mechanism. Examples of estrogen receptor        modulators include, among others, estrogens progestagen,        estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424,        tamoxifen, idoxifene, L Y353381, LY117081, toremifene,        fluvestrant,        4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate        4,4′dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH646.    -   Calcitonin: directly inhibits the osteoclast activity through        the calcitonin receptor. The calcitonin receptors have been        identified on the surface of the osteoclasts.    -   Bisphosphonates: are a group of medicinal products used for the        prevention and the treatment of diseases with bone resorption        and reabsorption such as osteoporosis and cancer with bone        metastasis, the latter being with or without hypercalcaemia,        associated to breast cancer and prostate cancer. Examples of        bisphosphonates which can be used in the therapy designed by        means of the fifth method of the invention include, although not        limited to, nitrogenous bisphosphonates (such as pamidronate,        neridronate, olpadronate, alendronate, ibandronate, risedronate,        incadronate, zoledronate or zoledronic acid, etc.) and        non-nitrogenous bisphosphonates (such as etidronate, clodronate,        tiludronate, etc.).    -   “Cathepsin K inhibitors” refers to compounds which interfere in        the cathepsin K cysteine protease activity. Non-limiting        examples of cathepsin K inhibitors include        4-amino-pyrimidine-2-carbonitrile derivatives (described in the        International patent application WO 03/020278 under the name of        Novartis Pharma GMBH), pyrrolo-pyrimidines described in the        publication WO 03/020721 (Novartis Pharma GMBH) and the        publication WO 04/000843 (ASTRAZENECA AB) as well as the        inhibitors described in the publications PCT WO 00/55126 of Axys        Pharmaceuticals, WO 01/49288 of Merck Frosst Canada & Co. and        Axys Pharmaceuticals.    -   “DKK-1(Dickkopf-1) inhibitor” as used herein refers to any        compound which is capable of reducing DKK-1 activity. DKK-1 is a        soluble Wnt pathway antagonist expressed predominantly in adult        bone and upregulated in myeloma patients with osteolytic        lesions. Agents targeting DKK-1 may play a role in preventing        osteolytic bone disease in multiple myeloma patients. BHQ880        from Novartis is a first-in-class, fully human, anti-DKK-1        neutralizing antibody. Preclinical studies support the        hypothesis that BHQ880 promotes bone formation and thereby        inhibits tumor-induced osteolytic disease (Ettenberg S. et al.,        American Association for Cancer Research Annual Meeting. Apr.        12-16, 2008; San Diego, Calif. Abstract).    -   “Dual MET and VEGFR2 inhibitor” as used herein refers to any        compound which is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. MET is        expressed not only in tumor cells and endothelial cells, but        also in osteoblasts (bone-forming cells) and osteoclasts        (bone-removing cells). HGF binds to MET on all of these cell        types, giving the MET pathway an important role in multiple        autocrine and paracrine loops. Activation of MET in tumor cells        appears to be important in the establishment of metastatic bone        lesions. At the same time, activation of the MET pathway in        osteoblasts and osteoclasts may lead to pathological features of        bone metastases, including abnormal bone growth (ie, blastic        lesions) or destruction (ie, lytic lesion. Thus, targeting the        MET pathway may be a viable strategy in preventing the        establishment and progression of metastatic bone lesions.        Cabozantinib (Exelixis, Inc), formerly known as XL184 (CAS        849217-68-1), is a potent dual inhibitor of the MET and VEGF        pathways designed to block MET driven tumor escape. In multiple        preclinical studies cabozantinib has been shown to kill tumor        cells, reduce metastases, and inhibit angiogenesis (the        formation of new blood vessels necessary to support tumor        growth). Another suitable dual inhibitors are E7050        (N-[2-Fluoro-4-({2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonylaminopyridin-4-yl}oxy)phenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide        (2R,3R)-tartrate) (CAS 928037-13-2) or Foretinib (also known as        GSK1363089, XL880, CAS 849217-64-7).    -   “RANKL inhibitors” as used herein refer to any compound which is        capable of reducing the RANK activity. RANKL is found on the        surface of the osteoblast membrane of the stroma and        T-lymphocyte cells, and these T-lymphocyte cells are the only        ones which have demonstrated the capacity for secreting it. Its        main function is the activation of the osteoclasts, cells        involved in the bone resorption. The RANKL inhibitors can act by        blocking the binding of RANKL to its receptor (RANK), blocking        the RANK-mediated signaling or reducing the expression of RANKL        by blocking the transcription or the translation of RANKL. RANKL        antagonists or inhibitors suitable for use in the present        invention include, without limitation:        -   a suitable RANK protein which is capable of binding RANKL            and which comprises the entire or a fragment of the            extracellular domain of a RANK protein. The soluble RANK may            comprise the signal peptide and the extracellular domain of            the murine or human RANK polypeptides, or alternatively, the            mature form of the protein with the signal peptide removed            can be used.        -   Osteoprotegerin or a variant thereof with RANKL-binding            capacity.        -   RANKL-specific antisense molecules        -   Ribozymes capable of processing the transcribed products of            RANKL        -   Specific anti-RANKL antibodies. “Anti-RANKL antibody or            antibody directed against RANKL” is understood herein as all            that antibody which is capable of binding specifically to            the ligand of the activating receptor for the nuclear factor            KB (RANKL) inhibiting one or more RANKL functions. The            antibodies can be prepared using any of the methods which            are known by the person skilled in the art. Thus, the            polyclonal antibodies are prepared by means of immunizing an            animal with the protein to be inhibited. The monoclonal            antibodies are prepared using the method described by            Kohler, Milstein et al. (Nature, 1975, 256: 495). Antibodies            suitable in the context of the present invention include            intact antibodies which comprise a variable antigen binding            region and a constant region, fragments “Fab”, “F(ab′)2” and            “Fab′”, Fv, scFv, diabodies and bispecific antibodies.        -   Specific anti-RANKL nanobodies. Nanobodies are            antibody-derived therapeutic proteins that contain the            unique structural and functional properties of            naturally-occurring heavy-chain antibodies. The Nanobody            technology was originally developed following the discovery            that camelidae (camels and llamas) possess fully functional            antibodies that lack light chains. The general structure of            nanobodies is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        -   wherein FR1 to FR4 are the framework regions 1 to 4 CDR1 to            CDR3 are the complementarity determining regions 1 to 3.            These heavy-chain antibodies contain a single variable            domain (VHH) and two constant domains (CH2 and CH3).            Importantly, the cloned and isolated VHH domain is a            perfectly stable polypeptide harbouring the full            antigen-binding capacity of the original heavy-chain            antibody. These newly discovered VHH domains with their            unique structural and functional properties form the basis            of a new generation of therapeutic antibodies which Ablynx            has named Nanobodies.

In one embodiment, the RANKL inhibitor is selected from the groupconsisting of a RANKL specific antibody, a RANKL specific nanobody andosteoprotegerin. In a specific embodiment, the anti-RANKL antibody is amonoclonal antibody. In a yet more specific embodiment, the anti-RANKLantibody is Denosumab (Pageau, Steven C. (2009). mAbs 1 (3): 210-215,CAS number 615258-40-7) (the entire contents of which are herebyincorporated by reference). Denosumab is a fully human monoclonalantibody which binds to RANKL and prevents its activation (it does notbind to the RANK receptor). Various aspects of Denosumab are covered byU.S. Pat. Nos. 6,740,522; 7,411,050; 7,097,834; 7,364,736 (the entirecontents of each of which are hereby incorporated by reference in theirentirety). In another embodiment, the RANKL inhibitor an antibody,antibody fragment, or fusion construct that binds the same epitope asDenosumab.

In a preferred embodiment, the anti-RANKL nanobody is any of thenanobodies as described in WO2008142164, (the contents of which areincorporated in the present application by reference). In a still morepreferred embodiment, the anti-RANKL antibody is the ALX-0141 (Ablynx).ALX-0141 has been designed to inhibit bone loss associated withpost-menopausal osteoporosis, reumatoid arthritis, cancer and certainmedications, and to restore the balance of healthy bone metabolism.

In a preferred embodiment, the agent preventing the bone degradation isselected from the group consisting of a bisphosphonate, a RANKLinhibitor, PTH and PTHLH inhibitor or a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, Radium-223, calcitonin, and a cathepsin K inhibitor. In amore preferred embodiment the agent preventing the bone degradation is abisphosphonate. In a yet more preferred embodiment, the bisphosphonateis the zoledronic acid.

In one embodiment, a CCR5 antagonist is administered to prevent orinhibit metastasis of the primary prostate cancer tumor to bone. In oneembodiment, the CCR5 antagonist is a large molecule. In anotherembodiment, the CCR5 antagonist is a small molecule. In someembodiments, the CCR5 antagonist is Maraviroc (Velasco-Veláquez, M. etal. 2012. CCR5Antagonist Blocks Metastasis of Basal Breast Cancer Cells.Cancer Research. 72:3839-3850.). In some embodiments, the CCR5antagonist is Vicriviroc. Velasco-Veláquez, M. et al. 2012.CCR5Antagonist Blocks Metastasis of Basal Breast Cancer Cells. CancerResearch. 72:3839-3850.). In some aspects, the CCR5 antagonist isAplaviroc (Demarest J. F. et al. 2005. Update on Aplaviroc: An HIV EntryInhibitor Targeting CCR5. Retrovirology 2(Suppl. 1): S13). In someaspects, the CCR5 antagonist is a spiropiperidine CCR5 antagonist.(Rotstein D. M. et al. 2009. Spiropiperidine CCR5 antagonists.Bioorganic & Medicinal Chemistry Letters. 19 (18): 5401-5406. In someembodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, D. R. 2009.HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4(2): 82-7).

In a preferred embodiment the dual MET and VEGFR2 inhibitor is selectedfrom the group consisting of Cabozantinib, Foretinib and E7050.

In another aspect, the treatment is an mTor inhibitor. In some aspects,the mTor inhibitor is a dual mTor/PI3kinase inhibitor. In some aspects,the mTor inhibitor is used to prevent or inhibit metastasis. In someaspects the mTor inhibitor is selected from the group consisting of:ABI009 (sirolimus), rapamycin (sirolimus), Abraxane (paclitaxel), Absorb(everolimus), Afinitor (everolimus), Afinitor with Gleevec, AS703026(pimasertib), Axxess (umirolimus), AZD2014, BEZ235, Biofreedom(umirolimus), BioMatrix (umirolimus), BioMatrix flex (umirolimus),CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUSNEICH(sirolimus), Curaxin CBLC102 (mepacrine), DE109 (sirolimus), DS3078,Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus), Femara(letrozole), Hocena (antroquinonol), INK128, Inspiron (sirolimus),IPI504 (retaspimycin hydrochloride), KRN951 (tivozanib), ME344, MGA031(teplizumab), MiStent SES (sirolimus), MKCl, Nobori (umirolimus),OSI027, OVI123 (cordycepin), Palomid 529, PF04691502, Promus Element(everolimus), PWT33597, Rapamune (sirolimus), Resolute DES(zotarolimus), RG7422, SAR245409, SF1126, SGN75 (vorsetuzumabmafodotin), Synergy (everolimus), Taltorvic (ridaforolimus), Tarceva(erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V(everolimus), Zomaxx (zotarolimus), Zortress (everolimus), ZotarolimusEluting Peripheral Stent MEDTRONIC (zotarolimus), AP23841, AP24170,ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371,EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602,Sirolimus PNP SAMYANG BIOPHARMACEUTICALS (sirolimus), TOP216, VLI27,VS5584, WYE125132, XL388, Advacan (everolimus), AZD8055, Cypher SelectPlus Sirolimus eluting Coronary Stent (sirolimus), Cypher Sirolimuseluting coronary stent (sirolimus), Drug Coated Balloon (sirolimus),E-Magic Plus (sirolimus), Emtor (sirolimus), Esprit (everolimus),Evertor (everolimus), HBF0079, LCP-Siro (sirolimus), Limus CLARIS(sirolimus), mTOR Inhibitor CELLZOME, Nevo Sirolimus eluting CoronaryStent (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus),ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus),Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (sirolimus),Sirolimus WATSON (sirolimus) Siropan (sirolimus), Sirova (sirolimus),Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON(tacrolimus), TAFA93, Temsirolimus ACCORD (temsirolimus), TemsirolimusSANDOZ (temsirolimus), TOP216, Xience Prime (everolimus), Xience V(everolimus). In a specific aspect the mTor inhibitor is Afinitor(everolimus)(http://www.afinitor.com/index.jsp?usertrack.filter_applied=true&Novald=4029462064338207963;last accessed Nov. 28, 2012). In another aspect, mTor inhibitors can beidentified through methods known in the art. (See, e.g., Zhou, H. et al.Updates of mTor inhibitors. 2010. Anticancer Agents Med. Chem. 10(7):571-81, which is herein incorporated by reference). In some aspects, themTor inhibitor is used to treat or prevent or inhibit metastasis in apatient with advanced prostate cancer. In some aspects, the mTorinhibitor is used in combination with a second treatment. In someaspects, the second treatment is any treatment described herein.

In another aspect, the treatment is a Src kinase inhibitor. In someaspects, the Src inhibitor is used to prevent or inhibit metastasis. Insome aspects, the Src kinase inhibitor is selected from the group:AZD0530 (saracatinib), Bosulif (bosutinib), ENMD981693, KD020, KX01,Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588,AZD0424, c-Src Kinase Inhibitor KISSEI, CU201, KX2361, SKS927, SRN004,SUNK706, TG100435, TG100948, AP23451, Dasatinib HETERO (dasatinib),Dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src Kinase InhibitorKINEX, VX680, (tozasertib lactate), XL228, and SUNK706. In someembodiments, the Src kinase inhibitor is dasatinib. In another aspect,Src kinase inhibitors can be identified through methods known in the art(See, e.g., Sen, B. and Johnson, F. M. Regulation of Src Family Kinasesin Human Cancers. 2011. J. Signal Transduction. 2011: 14 pages, which isherein incorporated by reference). In some aspects, the Src kinaseinhibitor is used to treat or prevent or inhibit metastasis in a patientthat is positive for the SRC-responsive signature (SRS). In someaspects, the Src kinase inhibitor is used to treat or prevent or inhibitmetastasis in a patient with advanced prostate cancer. In some aspects,the Src kinase inhibitor is used in combination with a second treatment.In some aspects, the second treatment is any treatment described herein.

In another aspect, the treatment is a COX-2 inhibitor. In some aspects,the COX-2 inhibitor is used to prevent or inhibit metastasis. In someaspects, the COX-2 inhibitor is selected from the group: ABT963,Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketorolactromethamine), BAY1019036 (aspirin), BAY987111 (diphenhydramine,naproxen sodium), BAY11902 (piroxicam), BCIBUCH001 (ibuprofen),Capoxigem (apricoxib), CS502, CS670 (pelubiprofen), Diclofenac HPBCD(diclofenac), Diractin (ketoprofen), GW406381, HCT1026(nitroflurbiprofen), Hyanalgese-D (diclofenac), HydrocoDex(acetaminophen, dextromethorphan, hydrocodone), Ibuprofen Sodium PFIZER(ibuprofen sodium), Ibuprofen with Acetaminophen PFIZER (acetaminophen,ibuprofen), Impracor (ketoprofen), IP880 (diclofenac), IP940(indomethacin), ISV205 (diclofenac sodium), JNS013 (acetaminophen,tramadol hydrochloride), Ketoprofen TDS (ketoprofen), LTNS001 (naproxenetemesil), Mesalamine SALIX (mesalamine), Mesalamine SOFAR (mesalamine),Mesalazine (mesalamine), ML3000 (licofelone), MRX7EAT (etodolac),Naproxen IROKO (naproxen), NCX4016 (nitroaspirin), NCX701(nitroacetaminophen), Nuprin SCOLR (ibuprofen), OMS103HP (amitriptylinehydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease(diclofenac), OxycoDex (dextromethorphan, oxycodone), P54, PercoDex(acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen,phosphatidyl choline), PSD508, R-Ketoprofen (ketoprofen), Remura(bromfenac sodium), ROX828 (ketorolac tromethamine), RP19583 (ketoprofenlysine), RQ00317076, SDX101 (R-etodolac), TDS943 (diclofenac sodium),TDT070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063(S-flurbiprofen), UR8880 (cimicoxib), V0498TA01A (ibuprofen), VT122(etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene),XP21B (diclofenac potassium), XP21L (diclofenac potassium), Zoenasa(acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed-P, Acular,Acular LS, Acular PF, Acular X, Acuvail, Advil, Advil Allergy Sinus,Advil Cold and Sinus, Advil Congestion Relief, Advil PM, Advil PMCapsule, Air Salonpas, Airtal, Alcohol-Free NyQuil Cold & Flu Relief,Aleve, Aleve ABDI IBRAHIM, Aleve-D, Alka-Seltzer, Alka-Seltzer BAYER,Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-SeltzerOriginal, Alka-Seltzer Plus, Alka-Seltzer plus Cold and Cough,Alka-Seltzer plus Cold and Cough Formula, Alka-Seltzer Plus Day andNight Cold Formula, Alka-Seltzer Plus Day Non-Drowsy Cold Formula,Alka-Seltzer Plus Flu Formula, Alka-Seltzer Plus Night Cold Formula,Alka-Seltzer Plus Sinus Formula, Alka-Seltzer Plus Sparkling OriginalCold Formula, Alka-Seltzer PM, Alka-Seltzer Wake-Up Call, Anacin,Anaprox, Anaprox MINERVA, Ansaid, Apitoxin, Apranax, Apranax abdi,Arcoxia, Arthritis Formula Bengay, Arthrotec, Asacol, Asacol HD, AsacolMEDUNA ARZNEIMITTEL, Asacol ORIFARM, Aspirin BAYER, Aspirin Complex,Aspirin Migran, AZD3582, Azulfidine, Baralgan M, BAY1019036, BAY987111,BAY11902, BCIBUCH001, Benadryl Allergy, Benadryl Day and Night, Benzylin4 Flu, Benzylin Cold and Flu, Benzylin Cold and Flu Day and Night,Benzylin Cold and Sinus Day and Night, Benzylin Cold and Sinus Plus,Benzylin Day and Night Cold and Flu Relief, Benzylinl All-In-One,Brexin, Brexin ANGELINI, Bromday, Bufferin, Buscopan Plus, Caldolor,Calmatel, Cambia, Canasa, Capoxigem, Cataflam, Celebrex, CelebrexORIFARM, Children's Advil Allergy Sinus, Children's Tylenol, Children'sTylenol Cough and Runny Nose, Children's Tylenol plus cold, Children'sTylenol plus Cold and Cough, Children's Tylenol plus cold and stuffynose, Children's Tylenol plus Flu, Children's Tylenol plus cold &allergy, Children's Tylenol plus Cough & Runny Nose, Children's Tylenolplus Cough & Sore Throat, Children's Tylenol plus multi symptom cold,Clinoril, Codral Cold and Flu, Codral Day and Night Day Tablets, CodralDay and Night Night Tablets, Codral Nightime, Colazal, Combunox, ContacCold plus Flu, Contac Cold plus Flu Non-Drowsy, Coricidin D, CoricidinHBP Cold and Flu, Coricidin HBP Day and Night Multi-Symptom Cold,Coricidin HBP Maximum Strength Flu, Coricidin HBP NighttimeMulti-Symptom Cold, Coricidin II Extra Strength Cold and Flu, CS502,CS670, Daypro, Daypro Alta, DDSO6C, Demazin Cold and Flu, Demazin Cough,Cold and Flu, Demazin day/night Cold and Flu, Demazin PE Cold and Flu,Demazin PE day/night Cold and Flu, Diclofenac HPBCD, Dimetapp DayRelief, Dimetapp Multi-Symptom Cold and Flu, Dimetapp Night Relief,Dimetapp Pain and Fever Relief, Dimetapp PE Sinus Pain, Dimetapp PESinus Pain plus Allergy, Dipentum, Diractin, Disprin Cold ‘n’ Fever,Disprin Extra, Disprin Forte. Disprin Plus, Dristan Cold, DristanJunior, Drixoral Plus, Duexis, Dynastat, Efferalgan, Efferalgan PlusVitamin C, Efferalgan Vitamin C, Elixsure IB, Excedrin Back and Body,Excedrin Migraine, Excedrin PM, Excedrin Sinus Headache, ExcedrinTension Headache, Falcol, Fansamac, Feldene, FeverAll, Fiorinal,Fiorinal with Codeine, Flanax, Flector Patch, Flucam, Fortagesic,Gerbin, Giazo, Gladio, Goody's Back and Body Pain, Goody's Cool Orange,Goody's Extra Strength, Goody's PM, Greaseless Bengay, GW406381,HCT1026, He Xing Yi, Hyanalgese-D, HydrocoDex, Ibuprofen Sodium PFIZER,Ibuprofen with, Acetaminophen PFIZER, Icy Hot SANOFI AVENTIS, Impracor,Indocin, Indomethacin APP PHARMA, Indomethacin MYLAN, Infants' Tylenol,IP880, IP940, Iremod, ISV205, JNS013, Jr. Tylenol, Junifen, JuniorStrength Advil, Junior Strength Motrin, Ketoprofen TDS, Lemsip Max,Lemsip Max All in One, Lemsip Max All Night, Lemsip Max Cold and Flu,Lialda, Listerine Mouth Wash, Lloyds Cream, Lodine, Lorfit P, Loxonin,LTNS001, Mersyndol, Mesalamine SALIX, Mesalamine SOFAR, Mesalazine,Mesasal GLAXO, Mesasal SANOFI, Mesulid, Metsal Heat Rub, Midol Complete,Midol Extended Relief, Midol Liquid Gels, Midol PM, Midol Teen Formula,Migranin COATED TABLETS, ML3000, Mobic, Mohrus, Motrin, Motrin Cold andSinus Pain, Motrin PM, Movalis ASPEN, MRX7EAT, Nalfon, Nalfon PEDINOL,Naprelan, Naprosyn, Naprosyn RPG LIFE SCIENCE, Naproxen IROKO, NCX4016,NCX701, NeoProfen LUNDBECK, Nevanac, Nexcede, Niflan, Norgesic MEDICIS,Novalgin, Nuprin SCOLR, Nurofen, Nurofen Cold and Flu, Nurofen MaxStrength Migraine, Nurofen Plus, Nuromol, NyQuil with Vitamin C, Ocufen,OMS103HP, Oralease, Orudis ABBOTT JAPAN, Oruvail, Osteluc, OxycoDex,P54, Panadol, Panadol Actifast, Paradine, Paramax, Parfenac, Pedea,Pennsaid, Pentasa, Pentasa ORIFARM, Peon, Percodan, Percodan-Demi,PercoDex, Percogesic, Perfalgan, PL2200, PL3100, Ponstel, Prexige,Prolensa, PSD508, R-Ketoprofen, Rantudil, Relafen, Remura, Robaxisal,Rotec, Rowasa, ROX828, RP19583, RQ00317076, Rubor, Salofalk, Salonpas,Saridon, SDX101, Seltouch, sfRowasa, Shinbaro, Sinumax, Sinutab,Sinutab, sinus, Spalt, Sprix, Strefen, Sudafed Cold and Cough, SudafedHead Cold and Sinus, Sudafed PE Cold plus Cough, Sudafed PE Pressureplus Pain, Sudafed PE, Severe Cold, Sudafed PE Sinus Day plus NightRelief Day Tablets, Sudafed PE Sinus Day plus Night Relief NightTablets, Sudafed PE Sinus plus Anti-inflammatory Pain Relief, SudafedSinus Advance, Surgam, Synalgos-DC, Synflex, Tavistallergy/sinus/headache, TDS943, TDT070, Theraflu Cold and Sore Throat,Theraflu Daytime Severe Cold and Cough, Theraflu Daytime WarmingRelief,Theraflu Warming Relief Caplets Daytime Multi-Symptom Cold,Theraflu Warming Relief Cold and Chest Congestion, Thomapyrin,Thomapyrin C, Thomapyrin Effervescent, Thomapyrin Medium, Tilcotil,Tispol, Tolectin, Toradol, TPR100, TQ1011, Trauma-Salbe, Trauma-SalbeKwizda, Treo, Treximet, Trovex, TT063, Tylenol, Tylenol AllergyMulti-Symptom, Tylenol Back Pain, Tylenol Cold & Cough Daytime, TylenolCold & Cough Nighttime, Tylenol Cold and Sinus Daytime, Tylenol Cold andSinus Nighttime, Tylenol Cold Head Congestion Severe, Tylenol Cold MultiSymptom Daytime, Tylenol Cold Multi Symptom Nighttime Liquid, TylenolCold Multi Symptom Severe, Tylenol Cold Non-Drowsiness Formula, TylenolCold Severe Congestion Daytime, Tylenol Complete Cold, Cough and FluNight time, Tylenol Flu Nighttime, Tylenol Menstrual, Tylenol PM,Tylenol Sinus Congestion & Pain Daytime, Tylenol Sinus Congestion & PainNighttime, Tylenol Sinus Congestion & Pain Severe, Tylenol Sinus SevereCongestion Daytime, Tylenol Ultra Relief, Tylenol with Caffeine andCodeine phosphate, Tylenol with Codeine phosphate, Ultra Strength BengayCream, Ultracet, UR8880, V0498TA01A, Vicks NyQuil Cold and Flu Relief,Vicoprofen, Vimovo, Voltaren Emulgel, Voltaren GEL, Voltaren NOVARTISCONSUMER HEALTH GMBH, Voltaren XR, VT122, Xefo, Xefo Rapid, Xefocam,Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, Zipsor, and Zoenasa. In anotheraspect, COX-2 inhibitors can be identified through methods known in theart (See, e.g., Dannhardt, G. and Kiefer, W. Cyclooxygenaseinhibitors-current status and future prospects. 2001. Eur. J. Med. Chem.36: 109-126, which is herein incorporated by reference). In someaspects, the COX-2 inhibitor is used to treat or prevent or inhibitmetastasis in a patient with advanced prostate cancer. In some aspects,the COX-2 inhibitor is used in combination with a second treatment. Insome aspects, the second treatment is any treatment described herein. Insome aspects, the COX-2 inhibitor is used in combination with a secondtreatment selected from the group consisting of: Denosumab, Zometa(http://www.us.zometa.com/index.jsp?usertrack.filter_applied=true&Novald=2935376934467633633; last accessed Dec. 2, 2012),Carbozantinib or Cabozantinib, Antibody or peptide blocking PTHLH(parathyroid hormone like hormone) or PTHrP (parathyroid hormone relatedprotein).

In one embodiment, the treatment is Radium 223. In a preferredembodiment the Radium 223 therapy is Alpharadin (aka, Xofigo)(radium-223 dichloride). Alpharadin uses alpha radiation from radium-223decay to kill cancer cells. Radium-223 naturally self-targets to bonemetastases by virtue of its properties as a calcium-mimic. Alpharadiation has a very short range of 2-10 cells (when compared to currentradiation therapy which is based on beta or gamma radiation), andtherefore causes less damage to surrounding healthy tissues(particularly bone marrow). With similar properties to calcium,radium-223 is drawn to places where calcium is used to build bone in thebody, including the site of faster, abnormal bone growth—such as thatseen in the skeletal metastases of men with advanced,castration-resistant prostate cancer. Radium-223, after injection, iscarried in the bloodstream to sites of abnormal bone growth. The placewhere a cancer starts in the body is known as the primary tumor. Some ofthese cells may break away and be carried in the bloodstream to anotherpart of the body. The cancer cells may then settle in that part of thebody and form a new tumor. If this happens it is called a secondarycancer or a metastasis. Most patients with late stage prostate cancersuffer the maximum burden of disease in their bones. The aim withradium-223 is to selectively target this secondary cancer. Anyradium-223 not taken-up in the bones is quickly routed to the gut andexcreted.

Alternatively a combined treatment can be carried out in which more thanone agent from those mentioned above are combined to treat and/orprevent the metastasis or said agents can be combined with othersupplements, such as calcium or vitamin D or with a hormone treatment.

Method of Diagnosis or Prognosis of Metastasis in Prostate Cancer Basedon Detecting the Amplification of the c-MAF Gene

In one aspect, the invention relates to an in vitro method for thediagnosis of metastasis in a subject with prostate cancer (hereinafter,fourth diagnosis method of the invention) and/or for the prognosis ofthe tendency to develop metastasis in a subject with prostate cancerwhich comprises determining if the c-MAF gene is amplified in a tumortissue sample of said subject; wherein if said gene is amplified withrespect to a control sample, then said subject has a positive diagnosisfor metastasis or a greater tendency to develop metastasis.

The terms “c-MAF gene”, “metastasis”, “tumor sample”, “prostate cancer”,“diagnosis of metastasis in a subject with prostate cancer”, “prognosisof the tendency to develop metastasis in a subject with prostatecancer”, “subject”, “patient”, “subject having a positive diagnosis ofmetastasis”, “subject having a greater tendency to develop metastasis”have been described in detail in the context of the first method of theinvention and are equally applicable to the fourth method of theinvention.

In a particular embodiment, the degree of amplification of the c-MAFgene can be determined by means of determining the amplification of achromosome region containing said gene. Preferably, the chromosomeregion the amplification of which is indicative of the existence ofamplification of the c-MAF gene is the locus 16q22-q24 which includesthe c-MAF gene. The locus 16q22-q24 is located in chromosome 16, in thelong arm of said chromosome and in a range between band 22 and band 24.This region corresponds in the NCBI database with the contigsNT_(—)010498.15 and NT_(—)010542.15. In another preferred embodiment,the degree of amplification of the c-MAF gene can be determined by meansof using a probe specific for said gene.

The fourth diagnosis/prognosis method of the invention comprises, in afirst step, determining if the c-MAF gene is amplified in a tumor sampleof a subject. To that end, the amplification of the c-MAF gene in thetumor sample is compared with respect to a control sample.

The term “amplification of a gene” as understood herein refers to aprocess through which various copies of a gene or of a gene fragment areformed in an individual cell or a cell line. The copies of the gene arenot necessarily located in the same chromosome. The duplicated region isoften called an “amplicon”. Normally, the amount of mRNA produced, i.e.,the gene expression level also increases in proportion to the copynumber of a particular gene.

In a particular embodiment, the fourth method of the invention for thediagnoses of metastasis in a subject with prostate cancer and/or for theprognosis of the tendency to develop metastasis in a subject withprostate cancer, comprises determining the c-MAF gene copy number in atumor sample of said subject and comparing said copy number with thecopy number of a control or reference sample, wherein if the c-MAF copynumber is greater with respect to the c-MAF copy number of a controlsample, then the subject has a positive diagnosis of metastasis or agreater tendency to develop metastasis.

The control sample refers to a tumor sample of a subject with prostatecancer who has not suffered metastasis or that correspond to the medianvalue of the c-MAF gene copy number measured in a tumor tissuecollection in biopsy samples of subjects with prostate cancer who havenot suffered metastasis. Said reference sample is typically obtained bycombining equal amounts of samples from a subject population. If thec-MAF gene copy number is increased with respect to the copy number ofsaid gene in the control sample, then subject has a positive diagnosisfor metastasis or a greater tendency to develop metastasis.

As used herein, the term “gene copy number” refers to the copy number ofa nucleic acid molecule in a cell. The gene copy number includes thegene copy number in the genomic (chromosomal) DNA of a cell. In a normalcell (non-tumoral cell), the gene copy number is normally two copies(one copy in each member of the chromosome pair). The gene copy numbersometimes includes half of the gene copy number taken from samples of acell population.

In the present invention, “increased gene copy number” is understood aswhen the c-MAF gene copy number is more than the copy number that areference sample or control sample has. In particular, it can beconsidered that a sample has an increased c-MAF copy number when thecopy number is more than 2 copies, for example, 3, 4, 5, 6, 7, 8, 9 or10 copies, and even more than 10 copies of the c-MAF gene.

In some embodiments, the amplification is in region at the 16q23 locus.In some embodiments, the amplification is in any part of the chromosomalregion between Chr. 16—79,392,959 bp to 79,663,806 bp (from centromereto telomere). In some embodiments, the amplification is in the genomicregion between Chr. 16—79,392,959 bp to 79,663,806 bp, but excluding DNArepeating elements. In some embodiments, amplification is measured usinga probe specific for that region.

In a particular embodiment, the amplification or the copy number isdetermined by means of in situ hybridization or PCR.

Methods for determining whether the c-MAF gene or the chromosome region16q22-q24 is amplified are widely known in the state of the art. Saidmethods include, without limitation, in situ hybridization (ISH) (suchas fluorescence in situ hybridization (FISH), chromogenic in situhybridization (CISH) or silver in situ hybridization (SISH)), genomiccomparative hybridization or polymerase chain reaction (such as realtime quantitative PCR). For any ISH method, the amplification or thecopy number can be determined by counting the number of fluorescentpoints, colored points or points with silver in the chromosomes or inthe nucleus.

The fluorescence in situ hybridization (FISH) is a cytogenetic techniquewhich is used for detecting and locating the presence or absence ofspecific DNA sequences in chromosomes. FISH uses fluorescence probeswhich only bind to some parts of the chromosome with which they show ahigh degree of sequence similarity. In a typical FISH method, the DNAprobe is labeled with a fluorescent molecule or a hapten, typically inthe form of fluor-dUTP, digoxigenin-dUTP, biotin-dUTP or hapten-dUTPwhich is incorporated in the DNA using enzymatic reactions, such as nicktranslation or PCR. The sample containing the genetic material (thechromosomes) is placed on glass slides and is denatured by a formamidetreatment. The labeled probe is then hybridized with the samplecontaining the genetic material under suitable conditions which will bedetermined by the person skilled in the art. After the hybridization,the sample is viewed either directly (in the case of a probe labeledwith fluorine) or indirectly (using fluorescently labeled antibodies todetect the hapten).

In the case of CISH, the probe is labeled with digoxigenin, biotin orfluorescein and is hybridized with the sample containing the geneticmaterial in suitable conditions.

Any marking or labeling molecule which can bind to a DNA can be used tolabel the probes used in the fourth method of the invention, thusallowing the detection of nucleic acid molecules. Examples of labels forthe labeling include, although not limited to, radioactive isotopes,enzyme substrates, cofactors, ligands, chemiluminescence agents,fluorophores, haptens, enzymes and combinations thereof. Methods forlabeling and guideline for selecting suitable labels for differentpurposes can be found, for example, in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wileyand Sons, New York, 1998).

Once the existence of amplification is determined, either by directlydetermining the amplification of the c-MAF gene or by determining theamplification of the locus 16q22-q24, and after being compared with theamplification of said gene in the control sample, if amplification inthe c-MAF gene is detected, it is indicative of the fact that thesubject has a positive diagnosis for metastasis or a greater tendency todevelop metastasis.

The determination of the amplification of the c-MAF gene needs to becorrelated with values of a control sample or reference sample thatcorrespond to the level of amplification of the c-MAF gene measured in atumor tissue sample of a subject with prostate cancer who has notsuffered metastasis or that correspond to the median value of theamplification of the c-MAF gene measured in a tumor tissue collection inbiopsy samples of subjects with prostate cancer who have not sufferedmetastasis. Said reference sample is typically obtained by combiningequal amounts of samples from a subject population. In general, thetypical reference samples will be obtained from subjects who areclinically well documented and in whom the absence of metastasis is wellcharacterized. The sample collection from which the reference level isderived will preferably be made up of subjects suffering the same typeof cancer as the patient object of the study. Once this median value hasbeen established, the level of amplification of c-MAF in tumor tissuesof patients can be compared with this median value, and thus, if thereis amplification, the subject has a positive diagnosis of metastasis ora greater tendency to develop metastasis.

In a preferred embodiment, the metastasis is bone metastasis. In a yetmore preferred embodiment, the bone metastasis is osteolytic bonemetastasis. As used herein, the expression “osteolytic bone metastasis”refers to a type of metastasis in which bone resorption (progressiveloss of bone density) is produced in the proximity of the metastasisresulting from the stimulation of the osteoclast activity by the tumorcells and is characterized by severe pain, pathological fractures,hypercalcaemia, spinal cord compression and other syndromes resultingfrom nerve compression.

Method of Prognosis of Metastasis in Prostate Cancer Based on Detectingthe Translocation of the c-MAF Gene

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering from prostatecancer, which comprises determining if the c-MAF gene is translocated ina sample of said subject wherein a translocation of the c-MAF gene isindicative of a poor clinical outcome.

In another aspect, the invention relates to an in vitro method forpredicting the clinical outcome of a patient suffering prostate cancer,which comprises determining if the c-MAF gene is translocated in asample of said subject wherein a translocation of the c-MAF gene isindicative of a poor clinical outcome.

In some embodiments, the translocated gene is from the region at the16q23 locus. In some embodiments, the translocated gene is from any partof the chromosomal region between Chr. 16-79,392,959 bp to 79,663,806 bp(from centromere to telomere). In some embodiments, the translocatedgene is from the genomic region between Chr. 16—79,392,959 bp to79,663,806 bp, but excluding DNA repeating elements. In someembodiments, the translocation is measured using a probe specific forthat region.

In a particular embodiment, the translocation of the c-MAF gene can bedetermined by means of determining the translocation of a chromosomeregion containing said gene. In one embodiment, the translocation is thet(14,16) translocation. In another embodiment, the chromosome regionthat is translocated is from locus 16q22-q24. The locus 16q22-q24 islocated in chromosome 16, in the long arm of said chromosome and in arange between band 22 and band 24. This region corresponds in the NCBIdatabase with the contigs NT_(—)010498.15 and NT_(—)010542.15. In apreferred embodiment, the c-MAF gene translocates to chromosome 14 atthe locus 14q32, resulting in the translocation t(14,16) (q32,q23). Thistranslocation places the MAF gene next to the strong enhancers in theIgH locus, which, in some cases, leads to overexpression of MAF.(Eychène, A., Rocques, N., and Puoponnot, C., A new MAFia in cancer.2008. Nature Reviews: Cancer. 8: 683-693.)

In a preferred embodiment, the translocation of the c-MAF gene can bedetermined by means of using a probe specific for said translocation. Insome embodiments, the translocation is measured using a dual colorprobe. In some embodiments, the translocation is measured using a dualfusion probe. In some embodiments, the translocation is measured using adual color, dual fusion probe. In some embodiments, the translocation ismeasured using two separate probes.

In another preferred embodiment, the translocation of the c-MAF gene isdetermined using the Vysis LSI IGH/MAF Dual Color dual fusion probe(http://www.abbottmolecular.com/us/products/analyte-specific-reagent/fish/vysis-lsi-igh-maf-dual-color-dual-fusion-probe.html;last accessed Nov. 5, 2012), which comprises a probe against 14q32 and16q23. In another preferred embodiment, the translocation of the c-MAFgene is determined using a Kreatech diagnostics MAF/IGH gt(14;16) Fusionprobe(http://www.kreatech.com/products/repeat-freetm-poseidontm-fish-probes/hematology/maf-igh-gtl416-fusion-probe.html;last accessed Nov. 5, 2012), an Abnova MAF FISH probe(http://www.abnova.com/products/products_detail.asp?Catalog _id=FA0375;last accessed Nov. 5, 2012), a Cancer Genetics Italia IGH/MAF Two Color,Two Fusion translocation probe(http://www.cancergeneticsitalia.com/dna-fish-probe/ighmaf/; lastaccessed Nov. 5, 2012), a Creative Bioarray IGH/MAF-t(14;16)(q32;q23)FISH probe(http://www.creative-bioarray.com/products.asp?cid=35&page=10; lastaccessed Nov. 5, 2012), a Arup Laboratories multiple myeloma panel byFISH (http://www.aruplab.com/files/technical-bulletins/Multiple%20Myeloma %20%28MM %29%20by %20FISH.pdf; last accessed Nov. 5, 2012),an Agilent probe specific to 16q23 or 14q32(http://www.genomics.agilent.com/ProductSearch.aspx?chr=16&start=79483700&end=79754340; last accessed Nov. 5, 2012;http://www.genomics.agilent.com/ProductSearch.aspx?Pageid=3000&ProductID=637; last accessed Nov. 5, 2012), a Dako probe specific to16q23 or 14q32(http://www.dako.com/us/ar42/psg42806000/baseproducts_surefish.htm?setCountry=true&purl=ar42/psg42806000/baseproducts_surefish.htm?undefined&submit=Accept%20country; last accessed Nov. 5, 2012), a Cytocell IGH/MAFTranslocation, Dual Fusion Probe(http://www.zentech.be/uploads/docs/products_info/prenatalo gy/cytocell%202012-2013%20catalogue %5B3%5D.pdf; last accessed Nov. 5, 2012), aMetasystems XL IGH/MAF Translocation—Dual Fusion Probe(http://www.metasystems-international.com/index.php?option=com_joodb&view=article&joobase=5&id=12%3Ad-5029-100-og&Itemid=272; last accessed Nov. 5, 2012),a Zeiss FISH Probes XL, 100 μl, IGH/MAFB(https://www.micro-shop.zeiss.com/?s=440675675dedc6&1=en&p=uk&f=r&i=5000&o=&h=25&n=1&sd=000000-0528-231-uk;last accessed Nov. 5, 2012) or a Genycell Biotech IGH/MAF Dual FusionProbe (http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCQQFjAA&url=http %3A %2F %2Fwww.genycell.es %2Fimage%2Fproductos %2Fbrochures %2Flphmie6_(—)86.ppt&ei=MhGYUOi3GKWH0QGlt4DoDw&usg=AFQjCNEqQMbT8vQGjJbi9riEf31VgoFTFQ&sig2=V5IS8juEMVHB18Mv2Xx_Ww; last accessed Nov. 5, 2012)

In some embodiments, the label on the probe is a fluorophore. In someembodiments, the fluorophore on the probe is orange. In someembodiments, the fluorophore on the probe is green. In some embodiments,the fluorophore on the probe is red. In some cases, the fluorophore onthe probe is yellow. In some embodiments, one probe is labeled with ared fluorophore, and one with a green fluorophore. In some embodiments,one probe is labeled with a green fluorophore and one with an orangefluorophore. In some cases, the fluorophore on the probe is yellow. Forinstance, if the MAF-specific probe is labeled with a red fluorophore,and the IGH-specific probe is labeled with a green fluorophore, if whiteis seen it indicates that the signals overlap and translocation hasoccurred.

In some embodiments, the fluorophore is SpectrumOrange. In someembodiments, the fluorophore is SpectrumGreen. In some embodiments, thefluorophore is DAPI. In some embodiments, the fluorophore isPlatinumBright405 In some embodiments, the fluorophore isPlatinumBright415. In some embodiments, the fluorophore isPlatinumBright495. In some embodiments, the fluorophore isPlatinumBright505. In some embodiments, the fluorophore isPlatinumBright550. In some embodiments, the fluorophore isPlatinumBright547. In some embodiments, the fluorophore isPlatinumBright570. In some embodiments, the fluorophore isPlatinumBright590. In some embodiments, the fluorophore isPlatinumBright647. In some embodiments, the fluorophore isPlatinumBright495/550. In some embodiments, the fluorophore isPlatinumBright415/495/550. In some embodiments, the fluorophore isDAPI/PlatinumBright495/550. In some embodiments, the fluorophore isFITC. In some embodiments, the fluorophore is Texas Red. In someembodiments, the fluorophore is DEAC. In some embodiments, thefluorophore is R6G. In some embodiments, the fluorophore is Cy5. In someembodiments, the fluorophore is FITC, Texas Red and DAPI. In someembodiments, a DAPI counterstain is used to visualize the translocation,amplification or copy number alteration.

One embodiment of the invention comprises a method in which in a firststep it is determined if the c-MAF gene is translocated in a sample of asubject. In a preferred embodiment, the sample is a tumor tissue sample.

In a particular embodiment, a method of the invention for the prognosisof the tendency to develop bone metastasis in a subject with prostatecancer comprises determining the c-MAF gene copy number in a sample ofsaid subject wherein the c-MAF gene is translocated and comparing saidcopy number with the copy number of a control or reference sample,wherein if the c-MAF copy number is greater with respect to the c-MAFcopy number of a control sample, then the subject has a greater tendencyto develop bone metastasis.

Methods for determining whether the c-MAF gene or the chromosome region16q22-q24 is translocated are widely known in the state of the art andinclude those described previously for the amplification of c-MAF. Saidmethods include, without limitation, in situ hybridization (ISH) (suchas fluorescence in situ hybridization (FISH), chromogenic in situhybridization (CISH) or silver in situ hybridization (SISH)), genomiccomparative hybridization or polymerase chain reaction (such as realtime quantitative PCR). For any ISH method, the amplification, the copynumber, or the translocation can be determined by counting the number offluorescent points, colored points or points with silver in thechromosomes or in the nucleus. In other embodiments, the detection ofcopy number alterations and translocations can be detected through theuse of whole genome sequencing, exome sequencing or by the use of anyPCR derived technology. For instance, PCR can be performed on samples ofgenomic DNA to detect translocation. In one embodiment, quantitative PCRis used. In one embodiment, PCR is performed with a primer specific tothe c-MAF gene and a primer specific to the IGH promoter region; if aproduct is produced, translocation has occurred.

In some embodiments, the amplification and copy number of the c-MAF geneare determined after translocation of the c-MAF gene is determined. Insome embodiments, the probe is used to determine if the cell ispolyploid for the c-MAF gene. In some embodiments, a determination ofpolyploidy is made by determining if there are more than 2 signals fromthe gene of interest. In some embodiments, polyploidy is determined bymeasuring the signal from the probe specific for the gene of interestand comparing it with a centromeric probe or other probe.

Method of Prognosis of Clinical Outcome in Prostate Cancer Based onDetecting the Amplification or Translocation of the c-MAF Gene

In another aspect, the invention relates to an in vitro method(hereinafter seventh method of the invention) for predicting theclinical outcome of a patient suffering prostate cancer, which comprisesdetermining if the c-MAF gene is amplified or translocated in a sampleof said subject relative to a reference gene copy number wherein anamplification of the c-MAF gene with respect to said reference gene copynumber is indicative of a poor clinical outcome.

The seventh method of the invention comprises, in a first step,determining if the c-MAF gene is amplified in a sample of a subject. Thedetermination of the amplification of the c-MAF is carried outessentially as described in the fifth method of the invention. In apreferred embodiment the sample is a tumor tissue sample. In a preferredembodiment, the amplification of the c-MAF gene is determined by meansof determining the amplification of the locus 16q23 or 16q22-q24. Inanother preferred embodiment, the amplification of the c-MAF gene isdetermined by means of using a c-MAF gene-specific probe.

In a second step, the seventh method of the invention comprisescomparing said copy number with the copy number of a control orreference sample, wherein if the c-MAF copy number is greater withrespect to the c-MAF copy number of a control sample, then this isindicative of a poor clinical outcome.

In a preferred embodiment, the c-MAF gene is amplified with respect to areference gene copy number when the c-MAF gene copy number is higherthan the copy number that a reference sample or control sample has. Inone example, the c-MAF gene is said to be “amplified” if the genomiccopy number of the c-MAF gene is increased by at least about 2- (i.e., 6copies), 3- (i.e., 8 copies), 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-,25-, 30-, 35-, 40-, 45-, or 50-fold in a test sample relative to acontrol sample. In another example, a c-MAF gene is said to be“amplified” if the genomic copy number of the c-MAF gene per cell is atleast about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and the like.

In another embodiment, the reference gene copy number is the gene copynumber in a sample of prostate cancer, from a subject who has notsuffered bone metastasis.

In another embodiment, the amplification is determined by means of insitu hybridization or PCR.

In another embodiment and as described in the present invention, giventhat the chr16q22-24, including the c-MAF gene, is amplified in prostatecancer cells is related to the presence of metastasis, the chr16q22-24,including the c-MAF gene, amplification allow making decisions in termsof the most suitable therapy for the subject suffering said cancer.

Thus, in another aspect the invention relates to an in vitro method fordesigning a customized therapy for a subject with prostate cancer, whichcomprises

-   -   (i) quantifying the chr16q22-24, including the c-MAF gene,        amplification in a tumor sample of said subject and    -   (ii) comparing the chr16q22-24, including the c-MAF gene,        amplification previously obtained with the degree of        amplification of said gene in a control sample,        wherein if the chr16q22-24, including the c-MAF gene, is        amplified with respect to the number of copies of said genomic        region in the control sample, then said subject is susceptible        to receive a therapy aiming to prevent and/or treat the        metastasis. In a particular aspect of this method, the subject        is then administered at least one therapeutic drug that        prevents, inhibits and/or treats the bone metastasis.        wherein if the chr16q22-24, including the c-MAF gene, is not        amplified with respect to the number of copies of said genomic        region in the reference sample, then said subject is not        susceptible to receive a therapy for preventing the bone        degradation. In a particular aspect of this method, the subject        is then not administered at least one therapeutic drug that        prevents, inhibits and/or treats the bone metastasis.

The invention relates to a therapeutic drug that prevents, inhibitsand/or treats the bone metastasis from those previously listed.

Therapeutic Methods of the Invention

Treating Bone Metastasis Using c-MAF Inhibitory Agents

A c-MAF gene expression inhibitory agent or an inhibitory agent of theprotein encoded by said gene can be used in the treatment and/or theprevention of prostate cancer metastasis.

Therefore, in another aspect, the invention relates to the use of ac-MAF gene expression inhibitory agent or an inhibitory agent of theprotein encoded by said gene (hereinafter, inhibitory agent of theinvention) in the preparation of a medicinal product for treating and/orpreventing prostate cancer metastasis. Alternatively, the inventionrelates to a c-MAF gene expression inhibitory agent or an inhibitoryagent of the protein encoded by said gene for use in the treatmentand/or the prevention of prostate cancer metastasis. Alternatively, theinvention relates to a method for treating the prostate cancermetastasis in a subject which comprises administering a c-MAF inhibitorto said subject. As used herein, a “c-MAF inhibitory agent” refers toany molecule capable of completely or partially inhibiting the c-MAFgene expression, both by preventing the expression product of said genefrom being produced (interrupting the c-MAF gene transcription and/orblocking the translation of the mRNA coming from the c-MAF geneexpression) and by directly inhibiting the c-MAF protein activity. C-MAFgene expression inhibitors can be identified using methods based on thecapacity of the so-called inhibitor to block the capacity of c-MAF topromote the in vitro cell proliferation, such as shown in theinternational patent application WO2005/046731 (hereby incorporated byreference in its entirety), based on the capacity of the so-calledinhibitor to block the transcription capacity of a reporter gene underthe control of the cyclin D2 promoter or of a promoter containing thec-MAF response region (MARE or c-MAF responsive element) in cells whichexpress c-MAF such as described in WO2008098351 (hereby incorporated byreference in its entirety) or based on the capacity of the so-calledinhibitor to block the expression of a reporter gene under the controlof the IL-4 promoter in response to the stimulation with PMA/ionomycinin cells which express NFATc2 and c-MAF such as described inUS2009048117A (hereby incorporated by reference in its entirety).

By way of non-limiting illustration, c-MAF inhibitory agents suitablefor use in the present invention include antisense oligonucleotides,interference RNAs (siRNAs), catalytic RNAs or specific ribozymes andinhibitory antibodies.

Antisense Oligonucleotides

An additional aspect of the invention relates to the use of isolated“antisense” nucleic acids to inhibit expression, for example, forinhibiting transcription and/or translation of a nucleic acid whichencodes c-MAF the activity of which is to be inhibited. The antisensenucleic acids can be bound to the target potential of the drug by meansof conventional base complementarity or, for example, in the case ofbinding to Double stranded DNA through specific interaction in the largegroove of the double helix. Generally, these methods refer to a range oftechniques generally used in the art and they include any method whichis based on the specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be administered, forexample, as an expression plasmid which, when is transcribed in cell,produces RNA complementary to at least one unique part of the cellularmRNA encoding c-MAF. Alternatively, the antisense construct is aoligonucleotide probe generated ex vivo which, when introduced into thecell, produces inhibition of gene expression hybridizing with the mRNAand/or gene sequences of a target nucleic acid. Such oligonucleotideprobes are preferably modified oligonucleotides which are resistant toendogenous nucleases, for example, exonucleases and/or endonucleases andare therefore stable in vivo. Examples of nucleic acid molecules for usethereof as an antisense oligonucleotides are DNA analogs ofphosphoramidate, phosphothionate and methylphosphonate (see also U.S.Pat. Nos. 5,176,996; 5,264,564; and 5,256,775) (hereby incorporated byreference in their entireties). Additionally, the general approximationsfor constructing oligomers useful in the antisense therapy have beenreviewed, for example, in Van der Krol et al., BioTechniques 6: 958-976,1988; and Stein et al., Cancer Res 48: 2659-2668, 1988.

With respect to the antisense oligonucleotide, theoligodeoxyribonucleotide regions derived from the starting site of thetranslation, for example, between −10 and +10 of the target gene arepreferred. The antisense approximations involve the oligonucleotidedesign (either DNA or RNA) that are complementary to the mRNA encodingthe target polypeptide. The antisense oligonucleotide will be bound tothe transcribed mRNA and translation will be prevented.

The oligonucleotides which are complementary to the 5′ end of the mRNA,for example the non translated 5′ sequence up to and including the startcodon AUG must function in the most efficient manner to inhibittranslation. Nevertheless, it has been shown that the sequencescomplementary to the non translated 3′ sequences of the mRNA are alsoefficient for inhibiting mRNA translation (Wagner, Nature 372: 333,1994). Therefore, complementary oligonucleotides could be used at thenon translated 5′ or 3′ regions, non coding regions of a gene in anantisense approximation to inhibit the translation of that mRNA. Theoligonucleotides complementary to the non translated 5′ region of themRNA must include the complement of the start codon AUG. Theoligonucleotides complementary to the coding region of the mRNA are lessefficient translation inhibitors but they could also be used accordingto the invention. If they are designed to hybridize with the 5′ region,3′ region or the coding region of the mRNA, the antisense nucleic acidsmust have at least about six nucleotides long and preferably have lessthan approximately 100 and more preferably less than approximately 50,25, 17 or 10 nucleotides long.

Preferably, in vitro studies are performed first to quantify thecapacity of the antisense oligonucleotides for inhibiting geneexpression. Preferably these studies use controls which distinguishbetween antisense gene inhibition and non specific biological effects ofthe oligonucleotides. Also preferably these studies compared the levelsof target RNA or protein with that of an internal control of RNA orprotein. The results obtained using the antisense oligonucleotides canbe compared with those obtained using a control oligonucleotide.Preferably the control oligonucleotide is approximately of the samelength as the oligonucleotide to be assayed and that the oligonucleotidesequence does not differ from the antisense sequence more than it isdeemed necessary to prevent the specific hybridization to the targetsequence.

The antisense oligonucleotide can be a single or double stranded DNA orRNA or chimeric mixtures or derivatives or modified versions thereof.The oligonucleotide can be modified in the base group, the sugar groupor the phosphate backbone, for example, to improve the stability of themolecule, its hybridization capacity etc. The oligonucleotide mayinclude other bound groups, such as peptides (for example, for directingthem to the receptors of the host cells) or agents for facilitatingtransport through the cell membrane (see, for example, Letsinger et al.,Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556, 1989; Lemaitre et al.,Proc. Natl. Acad. Sci. 84: 648-652, 1987; PCT Publication No. WO88/09810) or the blood-brain barrier (see, for example, PCT PublicationNo. WO 89/10134), intercalating agents (see, for example, Zon, Pharm.Res. 5: 539-549, 1988). For this purpose, the oligonucleotide can beconjugated to another molecule, for example, a peptide, a transportingagent, hybridization triggered cleaving agent, etc.

The antisense oligonucleotides may comprise at least one group ofmodified base. The antisense oligonucleotide may also comprise at leasta modified sugar group selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. The antisenseoligonucleotide may also contain a backbone similar to a neutralpeptide. Such molecules are known as peptide nucleic acid (PNA)oligomers and are described, for example, in Perry-O'Keefe et al., Proc.Natl. Acad. Sci. U.S.A. 93: 14670, 1996, and in Eglom et al., Nature365: 566, 1993.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone. In yet another embodiment, theantisense oligonucleotide is an alpha-anomeric oligonucleotide.

While antisense oligonucleotides complementary to the coding region ofthe target mRNA sequence can be used, those complementary to thetranscribed non translated region can also be used.

In some cases, it may be difficult to reach the sufficient intracellularconcentrations of the antisense to suppress the endogenous mRNAtranslation. Therefore, a preferred approximation uses a recombinant DNAconstruct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter.

Alternatively, the target gene expression can be reduced by directingdeoxyribonucleotide sequences complementary to the gene regulatingregion (i.e., the promoter and/or enhancers) to form triple helixstructures preventing gene transcription in the target cells in the body(see in general, Helene, Anticancer Drug Des. 6(6): 569-84, 1991). Incertain embodiments, the antisense oligonucleotides are antisensemorpholines.

siRNA

Small interfering RNA or siRNA are agents which are capable ofinhibiting the expression of a target gene by means of RNA interference.A siRNA can be chemically synthesized, can be obtained by means of invitro transcription or can be synthesized in vivo in the target cell.Typically, the siRNA consist of a double stranded RNA between 15 and 40nucleotides long and may contain a 3′ and/or 5′ protruding region of 1to 6 nucleotides. The length of the protruding region is independent ofthe total length of the siRNA molecule. The siRNA act by means ofdegrading or silencing the target messenger after transcription.

The siRNA of the invention are substantially homologous to the mRNA ofthe c-MAF encoding gene or to the gene sequence which encodes saidprotein. “Substantially homologous” is understood as having a sequencewhich is sufficiently complementary or similar to the target mRNA suchthat the siRNA is capable of degrading the latter through RNAinterference. The siRNA suitable for causing said interference includesiRNA formed by RNA, as well as siRNA containing different chemicalmodifications such as:

-   -   siRNA in which the bonds between the nucleotides are different        than those appearing in nature, such as phosphorothionate bonds.    -   Conjugates of the RNA strand with a functional reagent, such as        a fluorophore.    -   Modifications of the ends of the RNA strands, particularly of        the 3′ end by means of the modification with different hydroxyl        functional groups in 2′ position.    -   Nucleotides with modified sugars such as O-alkylated residues on        2′ position like 2′-O-methylribose or 2′-O-fluororibose.    -   Nucleotides with modified bases such as halogenated bases (for        example 5-bromouracil and 5-iodouracil), alkylated bases (for        example 7-methylguanosine).    -   The siRNA can be used as is, i.e., in the form of a double        stranded RNA with the aforementioned characteristics.        Alternatively, the use of vectors containing the sense and        antisense strand sequence of the siRNA is possible under the        control of suitable promoters for the expression thereof in the        cell of interest.    -   Vectors suitable for expressing siRNA are those in which the two        DNA regions encoding the two strands of siRNA are arranged in        tandem in one and the same DNA strand separated by a spacer        region which, upon transcription, forms a loop and wherein a        single promoter directs the transcription of the DNA molecule        giving rise to shRNA.    -   Alternatively, the use of vectors in which each of the strands        forming the siRNA is formed from the transcription of a        different transcriptional unit is possible. These vectors are in        turn divided into divergent and convergent transcription        vectors. In divergent transcription vectors, the transcriptional        units encoding each of the DNA strands forming the siRNA are        located in tandem in a vector such that the transcription of        each DNA strand depends on its own promoter which may be the        same or different (Wang, J. et al., 2003, Proc. Natl. Acad. Sci.        USA., 100:5103-5106 and Lee, N. S., et al., 2002, Nat.        Biotechnol., 20:500-505). In convergent transcription vectors,        the DNA regions giving rise to the siRNA form the sense and        antisense strands of a DNA region which are flanked by two        reverse promoters. After the transcription of the sense and        antisense RNA strands, the latter will form the hybrid for        forming a functional siRNA. Vectors with reverse promoter        systems in which 2 U6 promoters (Tran, N. et al., 2003, BMC        Biotechnol., 3:21), a mouse U6 promoter and a human H1 promoter        (Zheng, L., et al., 2004, Proc. Natl. Acad. Sci. USA., 135-140        and WO 2005026322) and a human U6 promoter and a mouse H1        promoter (Kaykas, A. and Moon, R., 2004, BMC Cell Biol., 5:16)        are used have been described.    -   Promoters suitable for use thereof in the expression of siRNA        from convergent or divergent expression vectors include any        promoter or pair of promoters compatible with the cells in which        the siRNA is to be expressed. Thus, promoters suitable for the        present invention include but are not necessarily limited to        constitutive promoters such as those derived from the genomes of        eukaryotic viruses such as the polyoma virus, adenovirus, SV40,        CMV, avian sarcoma virus, hepatitis B virus, the metallothionein        gene promoter, the thymidine kinase gene promoter of the herpes        simplex virus, retrovirus LTR regions, the immunoglobulin gene        promoter, the actin gene promoter, the EF-1alpha gene promoter        as well as inducible promoters in which the protein expression        depends on the addition of a molecule or an exogenous signal        such as the tetracycline system, the NFkappaB/UV light system,        the Cre/Lox system and the heat shock gene promoter, the        regulatable RNA polymerase II promoters described in        WO/2006/135436 as well as specific tissue promoters (for        example, the PSA promoter described in WO2006012221). In a        preferred embodiment, the promoters are RNA polymerase III        promoters which act constitutively. The RNA polymerase III        promoters are found in a limited number of genes such as 5S RNA,        tRNA, 7SL RNA and U6 snRNA. Unlike other RNA polymerase III        promoters, type III promoters do not require any intragenic        sequence but rather need sequences in 5′ direction comprising a        TATA box in positions −34 and −24, a proximal sequence element        or PSE between −66 and −47 and, in some cases, a distal sequence        element or DSE between positions −265 and −149. In a preferred        embodiment, the type III RNA polymerase III promoters are the        human or murine H1 and U6 gene promoters. In a yet more        preferred embodiment, the promoters are 2 human or murine U6        promoters, a mouse U6 promoter and a human H1 promoter or a        human U6 promoter and a mouse H1 promoter.    -   The siRNA can be generated intracellularly from the so called        shRNA (short hairpin RNA) characterized in that the antiparallel        strands forming the siRNA are connected by a loop or hairpin        region. The shRNAs can be encoded by plasmids or viruses,        particularly retroviruses, and are under the control of a        promoter. Promoters suitable for expressing shRNA are those        indicated in the paragraph above for expressing siRNA. Vectors        suitable for expressing siRNA and shRNA include prokaryotic        expression vectors such as pUC18, pUC19, Bluescript and the        derivatives thereof, mp18, mp19, pBR322, pMB9, CoIEl, pCRl, RP4,        phages and shuttle vectors such as pSA3 and pAT28, yeast        expression vectors such as 2-micron plasmid type vectors,        integration plasmids, YEP vectors, centromeric plasmids and the        like, insect cell expression vectors such as pAC series vectors        and pVL series vectors, plant expression vectors such as pIBI,        pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series        vectors and the like and viral vector-based (adenovirus, viruses        associated with adenoviruses as well as retroviruses and        particularly lentiviruses) higher eukaryotic cell expression        vectors or non-viral vectors such as pcDNA3, pHCMV/Zeo, pCR3.1,        pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV,        pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1,        pML2d and pTDTl. In a preferred embodiment, the vectors are        lentiviral vectors.    -   The siRNA and shRNA of the invention can be obtained using a        series of techniques known by the person skilled in the art. The        region of the nucleotide sequence taken as a basis for designing        the siRNA is not limiting and it may contain a region of the        coding sequence (between the start codon and the end codon) or        it may alternatively contain sequences of the non-translated 5′        or 3′ region preferably between 25 and 50 nucleotides long and        in any position in 3′ direction position with respect to the        start codon. One way of designing an siRNA involves the        identification of the AA(N19)TT motifs wherein N can be any        nucleotide in the c-MAF gene sequence, and the selection of        those having a high G/C content. If said motif is not found, it        is possible to identify the NA(N21) motif wherein N can be any        nucleotide.

c-MAF specific siRNAs include the siRNA described in WO2005046731(hereby incorporated by reference in its entirety), one of the strandsof which is ACGGCUCGAGCAGCGACAA (SEQ ID NO: 6). Other c-MAF specificsiRNA sequences include but are not limited to CUUACCAGUGUGUUCACAA (SEQID NO: 7), UGGAAGACUACUACUGGAUG (SEQ ID NO: 8), AUUUGCAGUCAUGGAGAACC(SEQ ID NO: 9), CAAGGAGAAAUACGAGAAGU (SEQ ID NO: 10),ACAAGGAGAAAUACGAGAAG (SEQ ID NO: 11) and ACCUGGAAGACUACUACUGG (SEQ IDNO: 12).

DNA Enzymes

On the other hand, the invention also contemplates the use of DNAenzymes to inhibit the expression of the c-MAF gene of the invention.DNA enzymes incorporate some of the mechanistic features of bothantisense and ribozyme technologies. DNA enzymes are designed such thatthey recognize a particular target nucleic acid sequence similar to theantisense oligonucleotide, nevertheless like the ribozyme they arecatalytic and specifically cleave the target nucleic acid.

Ribozymes

Ribozyme molecules designed for catalytically cleaving transcriptionproducts of a target mRNA to prevent the translation of the mRNA whichencodes c-MAF the activity of which is to be inhibited, can also beused. Ribozymes are enzymatic RNA molecules capable of catalyzingspecific RNA cleaving. (For a review, see, Rossi, Current Biology 4:469-471, 1994). The mechanism of ribozyme action involves a specifichybridization of a ribozyme molecule sequence to a complementary targetRNA followed by an endonucleolytic cleavage event. The composition ofthe ribozyme molecules preferably includes one or more sequencescomplementary to the target mRNA and the well known sequence responsiblefor cleaving the mRNA or a functionally equivalent sequence (see, forexample, U.S. Pat. No. 5,093,246).

The ribozymes used in the present invention include hammer-headribozymes and endoribonuclease RNA (hereinafter “Cech type ribozymes”)(Zaug et al., Science 224:574-578, 1984.

The ribozymes can be formed by modified oligonucleotides (for example toimprove the stability, targeting, etc.) and they should be distributedto cells expressing the target gene in vivo. A preferred distributionmethod involves using a DNA construct which “encodes” the ribozyme underthe control of a strong constitutive pol III or pol II promoter suchthat the transfected cells will produce sufficient amounts of theribozyme to destroy the endogenous target messengers and to inhibittranslation. Since the ribozymes are catalytic, unlike other antisensemolecules, a low intracellular concentration is required for itsefficiency.

Inhibitory Antibodies

In the context of the present invention, “inhibitory antibody” isunderstood as any antibody capable of binding specifically to the c-MAFprotein and inhibiting one or more of the functions of said protein,preferably those related to transcription. The antibodies can beprepared using any of the methods which are known by the person skilledin the art, some of which have been mentioned above. Thus, thepolyclonal antibodies are prepared by means of immunizing an animal withthe protein to be inhibited. The monoclonal antibodies are preparedusing the method described by Kohler, Milstein et al. (Nature, 1975,256: 495). In the context of the present invention, suitable antibodiesinclude intact antibodies comprising a variable antigen binding regionand a constant region, “Fab”, “F(ab′)2” and “Fab′”, Fv, scFv fragments,diabodies, bispecific antibodies, alphabodies, cyclopeptides and stapledpeptides. Once antibodies with c-MAF protein binding capacity areidentified, those capable of inhibiting the activity of this proteinwill be selected using an inhibitory agent identification assay.

Inhibitory Peptides

As used herein, the term “inhibitory peptide” refers to those peptidescapable of binding to the c-MAF protein and inhibiting its activity ashas been explained above, i.e., preventing the c-MAF from being able toactivate gene transcription.

Negative c-MAF Dominants

Since the proteins from the maf family are capable of homodimerizing andheterodimerizing with other members of the AP-1 family such as Fos andJun, one way of inhibiting c-MAF activity is by means of using negativedominants capable of dimerizing with c-MAF but lacking the capacity foractivating transcription. Thus, the negative c-MAF dominants can be anyof the small maf proteins existing in the cell and lacking two-thirds ofthe amino terminal end containing the transactivation domain (forexample, mafK, mafF, mafg and pi 8) (Fujiwara et al (1993) Oncogene 8,2371-2380; Igarashi et al. (1995) J. Biol. Chem. 270, 7615-7624; Andrewset al. (1993) Proc. Natl. Acad. Sci. USA 90, 11488-11492; Kataoka et al.(1995) Mol. Cell. Biol. 15, 2180-2190) (Kataoka et al. (1996) Oncogene12, 53-62).

Alternatively, the negative c-MAF dominants include c-MAF variants whichmaintain the capacity for dimerizing with other proteins but lack thecapacity for activating transcription. These variants are, for example,those lacking the c-MAF transactivation domain located at the N-terminalend of the protein. Thus, negative c-MAF dominant variants include in anillustrative manner the variants in which at least amino acids 1 to 122,at least amino acids 1-187 or at least amino acids 1 to 257 (byconsidering the numbering of human c-MAF as described in U.S. Pat. No.6,274,338, hereby incorporated by reference in its entirety) have beenremoved.

The invention contemplates the use of both the negative c-MAF dominantvariants and of polynucleotides encoding c-MAF under the operativecontrol of a promoter suitable for expression in target cell. Thepromoters that can be used for regulating the polynucleotidetranscription of the invention can be constitutive promoters, i.e.,promoters directing the transcription at a basal level, or induciblepromoters in which the transcriptional activity requires an externalsignal. Constitutive promoters suitable for regulating transcriptionare, among others, the CMV promoter, the SV40 promoter, the DHFRpromoter, the mouse mammary tumor virus (MMTV) promoter, the 1aelongation factor (EFla) promoter, the albumin promoter, the ApoA1promoter, the keratin promoter, the CD3 promoter, the immunoglobulinheavy or light chain promoter, the neurofilament promoter, the neuronspecific enolase promoter, the L7 promoter, the CD2 promoter, the myosinlight chain kinase promoter, the HOX gene promoter, the thymidine kinasepromoter, the RNA polymerase II promoter, the MyoD gene promoter, thephosphoglyceratekinase (PGK) gene promoter, the low density lipoprotein(LDL) promoter, the actin gene promoter. In a preferred embodiment, thepromoter regulating the expression of the transactivator is the PGK genepromoter. In a preferred embodiment, the promoter regulating thepolynucleotide transcription of the invention is the RNA polymerasepromoter of the T7 phage.

Preferably, the inducible promoters that can be used in the context ofthe present invention are those responding to an inducer agent showingzero or negligible basal expression in the absence of an inducer agentand are capable of promoting the activation of gene located in the 3′position. Depending on the type of inducer agent, the induciblepromoters are classified as Tet on/off promoters (Gossen, M. and H.Bujard (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551; Gossen, M. etal., 1995, Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau, 1998,Curr. Opin. Biotechnol. 9:451-456); Pip on/off promoters (U.S. Pat. No.6,287,813); antiprogestin-dependent promoters (US 2004132086),ecdysone-dependent promoters (Christopherson et al., 1992, Proc. Natl.Acad. Sci. USA, 89:6314-6318; No et al., 1996, Proc. Natl. Acad. Sci.USA, 93:3346-3351, Suhr et al., 1998, Proc. Natl. Acad. Sci. USA,95:7999-8004 and WO9738117), a metallothionein-dependent promoter(WO8604920) and rapamycin-dependent promoters (Rivera et al., 1996, Nat.Med. 2:1028-32).

Vectors suitable for expressing the polynucleotide encoding the negativec-MAF dominant variant include vectors derived from prokaryoticexpression vectors such as pUC18, pUC19, Bluescript and derivativesthereof, mp18, mp19, pBR322, pMB9, ColEl, pCRl, RP4, phages and shuttlevectors such as pSA3 and pAT28, yeast expression vectors such as2-micron type plasmid vectors, integration plasmids, YEP vectors,centromeric plasmids and the like, insect cell expression vectors suchas pAC series vectors and pVL series vectors, plant expression vectorssuch as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pOREseries vectors and the like and viral vector-based (adenoviruses,viruses associated with adenoviruses as well as retroviruses andparticularly lentiviruses) higher eukaryotic cell expression vectors ORnon-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3,pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL andpKSV-10, pBPV-1, pML2d and pTDTl.

Other Inhibitory Compounds of the c-MAF Protein Activity

Other c-MAF inhibitory compounds suitable for use in the presentinvention include:

TABLE 1 small molecules with c-MAF inhibiting capacity I Endiandric acidH derivatives such as those described in WO2004014888 corresponding tothe general formula

wherein R₁ and R₂ are, independently of one another, 1.0 H or 2.0 aO—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or —O—C₆-C₁₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups are mono-or disubstituted with: 2.1 —OH, 2.2 ═O, 2.3 —O—C₁-C6-alkyl, in whichalkyl is straight-chain or branched, 2.4 —O—C₂-C₆-alkenyl, in whichalkenyl is straight-chain or branched, 2.5 C₆-C₁₀-aryl, 2.6—NH—C₁-C₆-alkyl, in which alkyl is straight-chain or branched, 2.7—NH—C₂-C₆-alkenyl, in which alkenyl is straight-chain or branched, 2.8—NH₂ or 2.9 halogen, and in which the aryl group, is optionally mono- ordisubstituted with the substituent 2.1 or 2.3 to 2.9, in which thesubstituents 2.3, 2.4, 2.6 and 2.7 may be further substituted with —CN,-amide or -oxime functions, and 2.5 may be further substituted with —CNor amide functions, or R₁ and R₂ together form a ring, wherein R₁ and R₂mean a —O—[(C₁-C₆)-alkylene]—O— group, R₃ is 1.0 H or 2.0 a—O—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or —O—C₆-C₁₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups are mono-or disubstituted with: 2.1 —OH, 2.2 ═O, 2.3 —O—C₁-C₆-alkyl, in whichalkyl is straight-chain or branched, 2.4 —O—C₂-C₆-alkenyl, in whichalkenyl is straight-chain or branched, 2.5 —C₆-C₁₀-aryl, 2.6—NH—C₁-C₆-alkyl, in which alkyl is straight-chain or branched, 2.7—NH—C₂-C₆-alkenyl, in which alkenyl is straight-chain or branched, 2.8—NH₂ or 2.9 halogen, and in which the aryl group, is optionally mono- ordisubstituted with the substituent 2.1 or 2.3 to 2.9, in which thesubstituents 2.3, 2.4, 2.6 and 2.7 may be further substituted with —CN,-amide or -oxime functions, and 2.5 may be further substituted with —CNor amide functions R₄ is CO₂R₃, CO₂NHR₃, CHO, CH₂OR₃, CH₂OSi(R₃)₃,CH₂Br, CH₂CN, in which R₃ is as defined above, and, in particular, thecompounds

II 8-hydroxyquinoline derivatives such as those described inWO2009146546 of general formula

wherein R₁ is selected from the group consisting of NO₂, NH₂, NH(C₁-C₆-alkyl) and N(C₁-C₆-alkyl) (C₁-C₆-alkyl); R₂ is selected from H,halogen, C₁-C₆ alkyl, and fluoro- substituted C₁-C₆ alkyl, or R₁ is Cland R₂ is Br or H, and, preferably, the compounds

III Clioquinol (5-chloro-7-iodoquinolin-8-ol) as described in WO09049410IV Compounds such as those described in WO08098351 of general formula

wherein ═ ═—:—:—: is a single or double bond, R¹ is selected from thegroup consisting of H, (C₁-C₄ alkyl, C(O)O C₁-C₄ alkyl, C(O) C₁-C₄ alkyland C(O)NH C₁-C₄ alkyl; R² is selected from H and C₁-C₄ alkyl; R³ isselected from H and C₁-C₄ alkyl; or R² and R³ are bound together alongwith the carbon and nitrogen atoms to which they are bound to form apiperidine ring, R⁴ and R⁵ are independently selected from H, halogen,hydroxy, C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl and C₁- C₄ alkoxy;and X is selected from C and N, and preferred compounds such asCyproheptadine (4-(5H-dibenzo-[a,d]cyclohepten-5-ylidene)-1-methylpiperidine hydrochloride), Amitriptyline (3-(10,11-dihydro-5H-dibenzo[[a,d]]cycloheptene-5-ylidene)-N,N-dimethyl-1- propanamine),Loratadine (Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1- piperidinecarboxylate,Cyclobenzrapine (3-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-1-propanamine). V Nivalenol(12,13-epoxy-3,4,7,15-tetrahydroxytrichothec-9- en-8-one) as describedin WO0359249

Other c-MAF inhibitors are described in the patent applicationWO2005063252, such as shown in the following table (Table 2).

TABLE 2 c-MAF inhibitors Antagonist Reference for cdk2 inhibitoryactivity Purine Analogs Purvalanols such as 2-(1R-Isopropyl-2- Gray, N.S. et al., Science, 281, 533-538hydroxyethylamino)-6-(3-chloroanilino)-9- (1998); isopropylpurine havinga molecular formula Chang, Y. T. et al., Chem. Biol., 6, 361-375C₁₉H₂₅ClN₆O available from Sigma-Aldrich under (1999). the trade namePurvalanol A (#P4484, Sigma- Aldrich, St. Louis, MO), Purvalanol B,aminopurvalanol, compound 52 (where isopropyl of purvalanol A isreplaced with H) 2-(Hydroxyethylamino)-6-benzylamino-9- Vesely, J., etal., (1994) Eur. J. Biochem., 224, methylpurine having a molecularformula 771-86, 11; C₁₅H₁₈N₆O available from Sigma-Aldrich under Brooks,E. E., et al., (1997) J. Biol. Chem., 272, the trade name Olomoucine(#O0886), 29207-11 2-(2′-Hydroxyethylamino)-6-benzylamino-9-isopropylpurine having a molecular formula C₁₇H₂₂N₆O available fromSigma-Aldrich under the trade name N⁹-isopropylolomoucine (#I0763);CVT-313 6-(Benzylamino)-2(R)-[[1- Wang, D. et al., J. Virol., 75,7266-7279 (hydroxymethyl)propyl]amino]-9-isopropylpurine (2001); McClue,S. J. et al., Int. J. Cancer, 102, 2-(R)-[[9-(1-methylethyl)-6- 463-468(2002); [(phenylmethyl)amino]-9H-purin-2-yl]amino]-1- Meijer, L., etal., (1997) Eur. J. Biochem., 243, butanol having a molecular formula ofC₁₉H₂₆N₆O 527-36 available from Sigma-Aldrich under the trade nameRoscovitine (#R7772), methoxyroscovitine Purine analogN2-(cis-2-Aminocyclohexyl)-N6- Imbach, P. et al., Bioorg. Med. Chem.Lett., 9, (3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine 91-96 (1999);having a molecular formula of C₁₉H₂₄ClN₇ Dreyer, M. K. et al., J. Med.Chem., 44, 524-530 available from Sigma-Aldrich under the trade (2001).name CGP74514 (#C3353) CGP79807, a purine analog of CGP74514 (supra)Imbach, P. et al., Bioorg. Med. Chem. Lett., 9, where Cl is replacedwith CN, OH is removed, 91-96 (1999); and the ortho position ofcyclohexane ring is NH₂ Dreyer, M. K. et al., J. Med. Chem., 44, 524-530(2001). purine analog such as O6-cyclohexylmethyl Arris, C. E. et al.,J. Med. Chem., 43, 2797-2804 guanine NU2058 (2000); Davies et al, NatureStructural Biology, 9:10, 745-749, 2002 purine analog such as NU6102Arris, C. E. et al., J. Med. Chem., 43, 2797-2804 (2000); Davies, T. G.et al., Nat. Struct. Biol., 9, 745-749 (2002). isopentenyl-adenineVesely, J., et al., (1994) Eur. J. Biochem., 224, 771-86 Nonpurine basedagents Indirubins such as indirubin-3′-monoxime having Davies, T. G. etat., Structure, 9, 389-397 a molecular formula of C₁₆H₁₁N₃O₂ availablefrom (2001); Sigma-Aldrich under the trade name (#I0404), Marko, D. etal., Br. J. Cancer, 84, 283-289 indirubin 5-sulfonate, 5-chloroindirubin (2001); Hoessel, R., et al., (1999) Nat. Cell Biol., 1, 60-7;PCT/US02/30059 to Hellberg et al., published as WO 03/027275. Oxindole 1of Fischer as referenced in column 2 Porcs-Makkay, M., et al.,Tetrahedron 2000, of this table, (#IN118, JMAR Chemical, 56, 5893; Org.Process Res. Dev. 2000, 4, 10 Indenopyrazoles Nugiel, D. A. et al., J.Med. Chem., 44, 1334-1336 (2001); Nugiel, D. A. et al., J. Med. Chem.,45, 5224-5232 (2002); Yue, E. W. et al., J. Med. Chem., 45, 5233-5248(2002). Pyrido(2,3-d)pyrimidine-7-ones, compound 3 of Barvian, M. etal., J. Med. Chem., 43, 4606-4616 Fischer (2000); Toogood, P. L., Med.Res. Rev., 21, 487-498 (2001). Quinazolines such as anilinoquinazolineSielecki, T. M. et al., Bioorg. Med. Chem. Lett., 11, 1157-1160 (2001);Mettey et al., J. Med. Chem. 2003, 46, 222-236. Thiazoles such as fusedthiazole, 4-{[(7-Oxo-6,7- Davis, S. T. et al., Science, 291, 134-137dihydro-8H-[1,3]thiazolo[5,4-e]indol-8- (2001);ylidene)methyl]amino}-N-(2- PCT/US02/30059 to Hellberg et al., publishedpyridyl)benzenesulfonamide having a molecular as WO 03/027275. formulaof C₂₁H₁₅N₅O₃S₂ available from Sigma- Aldrich under the trade nameGW8510 (#G7791) Flavopiridols such as flavopiridol (L86 8275; Carlson,B. A., et al., (1996) Cancer Res., 56, NCS 649890, National CancerInstitute, Bethesda, 2973-8 MD) and a dechloro derivative Alkaloids suchas Staurosporine (#S1016, A.G. Rialet, V., et al., (1991) AnticancerRes., 11, Scientific, San Diego, CA) or UCN-01 (7- 1581-90;hydroxystaurosporine) National Cancer Institute, Wang, Q., et al.,(1995) Cell Growth Differ., 6, Bethesda, MD 927-36, Akiyama, T., et al.,(1997) Cancer Res., 57, 1495-501, Kawakami, K., et al., (1996) Biochem.Biophys. Res. Commun., 219, 778-83 Paullones such as9-Bromo-7,12-dihydro- Zaharevitz, D. W. et al., Cancer Res., 59,2566-2569 indolo[3,2-d][1]benzazepin-6(5H)-one having a (1999); Schultz,C. et al., J. Med. Chem., molecular formula of C₁₆H₁₁BrN₂O availablefrom 42, 2909-2919 (1999); Sigma-Aldrich under the trade namekenpaullone Zaharevitz, D. W., et al., (1999) Cancer Res., (#K3888), or9-Nitro-7,12-dihydroindolo-[3,2- 59, 2566-9; d][1]benzazepin-6(5)-onehaving a molecular PCT/US02/30059 to Hellberg et al., published formulaof C₁₆H₁₁N₃O₃ available from Sigma- as WO 03/027275. Aldrich under thetrade name alsterpaullone (#A4847) CGP 41251, an alkaloid Begemann, M.,et al., (1998) Anticancer Res., 18, 2275-82; Fabbro et al., PharmacolTher. 1999 May-Jun; 82(2-3): 293-301 Hymenialdisines such as10z-hymenialdisine Meijer, L., et al., (1999) Chemistry & Biology,having a molecular formula of C₁₁H₁₀BrN₅O₂ 7, 51-63; available fromBiochemicals.net, a division of PCT/US02/30059 to Hellberg et al.,published A.G. Scientific, Inc. (San Diego, CA) (H-1150) as WO03/027275. CGP60474, a phenylaminopyrimidine 21; WO95/09853, Zimmermannet al., Sep. 21, 1994 Thiazolopyrimidine 2 Attaby et al., Z.Naturforsch. 54b, 788-798 (1999) Diarylurea Honma, T. et al., J. Med.Chem., 44, 4628-4640 (2001), Honma, T. et al., J. Med. Chem., 44,4615-4627 (2001). (2R)-2,5-Dihydro-4-hydroxy-2-[(4-hydroxy-3-(3-Kitagawa, M. et al., Oncogene, 8, 2425-2432methyl-2-butenyl)phenyl)methyl]-3-(4- (1993).hydroxyphenyl)-5-oxo-2-furancarboxylic acid methyl ester having amolecular formula of C₂₄H₂₄O₇ available from Sigma-Aldrich under thetrade name Butyrolactone-I (B7930) Aloisine A, Cat. No. 128125(Calbiochem, San Mettey et al., J. Med. Chem. 2003, 46, 222-236 Diego,CA)

In a preferred embodiment, the c-MAF inhibitory agents are used for thetreatment and/or prevention of bone metastasis. In a yet more preferredembodiment, the bone metastasis is osteolytic metastasis.

The c-MAF inhibitory agents are typically administered in combinationwith a pharmaceutically acceptable carrier.

The term “carrier” refers to a diluent or an excipient whereby theactive ingredient is administered. Such pharmaceutical carriers can besterile liquids such as water and oil, including those of a petroleum,animal, plant or synthetic origin such peanut oil, soy oil, mineral oil,sesame oil and the like. Water or aqueous saline solutions and aqueousdextrose and glycerol solutions, particularly for injectable solutions,are preferably used as carriers. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin,1995. Preferably, the carriers of the invention are approved by thestate or federal government regulatory agency or are listed in theUnited States Pharmacopeia or other pharmacopeia generally recognizedfor use thereof in animals and more particularly in human beings.

The carriers and auxiliary substances necessary for manufacturing thedesired pharmaceutical dosage form of the pharmaceutical composition ofthe invention will depend, among other factors, on the pharmaceuticaldosage form chosen. Said pharmaceutical dosage forms of thepharmaceutical composition will be manufactured according to theconventional methods known by the person skilled in the art. A review ofthe different methods for administering active ingredients, excipientsto be used and processes for producing them can be found in “Tratado deFarmacia Galénica”, C. Faulí i Trillo, Luzan 5, S. A. 1993 Edition.Examples of pharmaceutical compositions include any solid composition(tablets, pills, capsules, granules, etc.) or liquid composition(solutions, suspensions or emulsions) for oral, topical or parenteraladministration. Furthermore, the pharmaceutical composition may contain,as deemed necessary, stabilizers, suspensions, preservatives,surfactants and the like.

For use in medicine, the c-MAF inhibitory agents can be found in theform of a prodrug, salt, solvate or clathrate, either isolated or incombination with additional active agents and can be formulated togetherwith a pharmaceutically acceptable excipient. Excipients preferred foruse thereof in the present invention include sugars, starches,celluloses, rubbers and proteins. In a particular embodiment, thepharmaceutical composition of the invention will be formulated in asolid pharmaceutical dosage form (for example tablets, capsules, pills,granules, suppositories, sterile crystal or amorphous solids that can bereconstituted to provide liquid forms etc.), liquid pharmaceuticaldosage form (for example solutions, suspensions, emulsions, elixirs,lotions, ointments etc.) or semisolid pharmaceutical dosage form (gels,ointments, creams and the like). The pharmaceutical compositions of theinvention can be administered by any route, including but not limited tothe oral route, intravenous route, intramuscular route, intraarterialroute, intramedularry route, intrathecal route, intraventricular router,transdermal route, subcutaneous route, intraperitoneal route, intranasalroute, enteric route, topical route, sublingual route or rectal route. Areview of the different ways for administering active ingredients, ofthe excipients to be used and of the manufacturing processes thereof canbe found in Tratado de Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A., 1993 Edition and in Remington's Pharmaceutical Sciences (A. R.Gennaro, Ed.), 20^(th) edition, Williams & Wilkins PA, USA (2000).Examples of pharmaceutically acceptable carriers are known in the stateof art and include phosphate buffered saline solutions, water, emulsionssuch as oil/water emulsions, different types of wetting agents, sterilesolutions, etc. The compositions comprising said carriers can beformulated by conventional processes known in the state of the art.

In the event that nucleic acids (siRNA, polynucleotides encoding siRNAor shRNA or polynucleotides encoding negative c-MAF dominants) areadministered the invention contemplates pharmaceutical compositionsparticularly prepared for administering said nucleic acids. Thepharmaceutical compositions can comprise said naked nucleic acids, i.e.,in the absence of compounds protecting the nucleic acids fromdegradation by the nucleases of the body, which entails the advantagethat the toxicity associated with the reagents used for transfection iseliminated. Administration routes suitable for naked compounds includethe intravascular route, intratumor route, intracranial route,intraperitoneal route, intrasplenic route, intramuscular route,subretinal route, subcutaneous route, mucosal route, topical route andoral route (Templeton, 2002, DNA Cell Biol., 21:857-867). Alternatively,the nucleic acids can be administered forming part of liposomesconjugated to cholesterol or conjugated to compounds capable ofpromoting the translocation through cell membranes such as the Tatpeptide derived from the HIV-1 TAT protein, the third helix of thehomeodomain of the D. melanogaster antennapedia protein, the herpessimplex virus VP22 protein, arginine oligomers and peptides as describedin WO07069090 (Lindgren, A. et al., 2000, Trends Pharmacol. Sci,21:99-103, Schwarze, S. R. et al., 2000, Trends Pharmacol. Sci.,21:45-48, Lundberg, M et al., 2003, Mol Therapy 8:143-150 and Snyder, E.L. and Dowdy, S. F., 2004, Pharm. Res. 21:389-393). Alternatively, thepolynucleotide can be administered forming part of a plasmid vector orviral vector, preferably adenovirus-based vectors, in adeno-associatedviruses or in retroviruses such as viruses based on murine leukemiavirus (MLV) or on lentivirus (HIV, FIV, EIAV).

The c-MAF inhibitory agents or the pharmaceutical compositionscontaining them can be administered at a dose of less than 10 mg perkilogram of body weight, preferably less than 5, 2, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight. The unit dose can be administered by injection, inhalation ortopical administration.

The dose depends on the severity and the response of the condition to betreated and it may vary between several days and months or until thecondition subsides. The optimal dosage can be determined by periodicallymeasuring the concentrations of the agent in the body of the patient.The optimal dose can be determined from the EC50 values obtained bymeans of previous in vitro or in vivo assays in animal models. The unitdose can be administered once a day or less than once a day, preferablyless than once every 2, 4, 8 or 30 days. Alternatively, it is possibleto administer a starting dose followed by one or several maintenancedoses, generally of a lesser amount than the starting dose. Themaintenance regimen may involve treating the patient with a dose rangingbetween 0.01 pg and 1.4 mg/kg of body weight per day, for example 10, 1,0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day. Themaintenance doses are preferably administered at the most once every 5,10 or 30 days. The treatment must be continued for a time that will varyaccording to the type of disorder the patient suffers, the severitythereof and the condition of the patient. After treatment, the progressof the patient must be monitored to determine if the dose should beincreased in the event that the disease does not respond to thetreatment or the dose is reduced if an improvement of the disease isobserved or if unwanted side effects are observed.

Treatment or Prevention of the Bone Degradation in Prostate CancerPatients with Bone Metastasis Having Elevated c-MAF Levels

Patients suffering prostate cancer which has metastasized in bone and inwhich there are elevated c-MAF levels in said metastasis may benefitparticularly from therapies aimed at preventing the bone degradationcaused by the increased osteoclastic activity.

Thus, in another aspect, the invention relates to the use of an agentfor avoiding or preventing bone degradation in the preparation of amedicinal product for the prevention and/or the treatment of the bonemetastasis in a subject suffering prostate cancer and having elevatedc-MAF levels in a metastatic tumor tissue sample with respect to acontrol sample.

Alternatively, the invention relates to an agent for avoiding orpreventing bone degradation for use in the prevention and/or thetreatment of the bone metastasis in a subject suffering prostate cancerand has elevated c-MAF levels in a metastatic tumor tissue sample withrespect to a control sample.

Alternatively, the invention relates to a method of prevention and/ortreatment of the degradation in a subject suffering prostate cancer andhas elevated c-MAF levels in a metastatic tumor tissue sample withrespect to a control sample, which comprises administering an agent foravoiding or preventing bone degradation to said subject.

In a particular embodiment, the bone metastasis is osteolyticmetastasis.

The terms and expressions “subject”, “prostate cancer”, “tumor sample”,“metastasis”, “c-MAF gene”, “increased or elevated expression levels”and “control sample” have been described in detail in relation with thefirst method of the invention and are equally applicable to the agentfor avoiding or preventing bone degradation.

Agents capable of avoiding or preventing bone degradation suitable forthe therapeutic method described in the present invention have beendescribed in detail above in the context of the customized therapymethod.

The reference or control sample is a tumor sample of a subject withprostate cancer who has not suffered metastasis or that corresponds tothe median value of the c-MAF gene expression levels measured in a tumortissue collection in biopsy samples of subjects with prostate cancer whohave not suffered metastasis.

Methods for determining or quantifying if the c-MAF levels are elevatedwith respect to a control sample have been described in detail inrelation with the first method of the invention and are equallyapplicable to the agent for avoiding or preventing bone degradation.

Alternatively a combined treatment can be carried out, in which morethan one agent for avoiding or preventing bone degradation from thosementioned above are combined to treat and/or prevent the metastasis orsaid agents can be combined with other supplements, such as calcium orvitamin D or with a hormone.

The agents for avoiding or preventing bone degradation are typicallyadministered in combination with a pharmaceutically acceptable carrier.The term “carrier” and the types of carriers have been defined above forthe c-MAF inhibitory agent, as well as the form and the dose in whichthey can be administered and are equally applicable to the agent foravoiding or preventing bone degradation.

The following examples illustrate the invention and do not limit thescope thereof.

Kits of the Invention

In another aspect, the invention relates to a kit for predicting bonemetastasis of a prostate cancer, in a subject suffering from saidcancer, the kit comprising: a) means for quantifying the expressionlevel of c-MAF in a sample of said subject; and b) means for comparingthe quantified level of expression of c-MAF in said sample to areference c-MAF expression level.

In another aspect, the invention relates to a kit for predicting theclinical outcome of a subject suffering from bone metastasis from aprostate cancer, the kit comprising: a) means for quantifying theexpression level of c-MAF in a sample of said subject; and b) means forcomparing the quantified expression level of c-MAF in said sample to areference c-MAF expression level.

In another aspect the invention relates to a kit for determining atherapy for a subject suffering from prostate cancer, the kitcomprising: a) means for quantifying the expression level of c-MAF in asample of said subject; b) means for comparing the quantified expressionlevel of c-MAF in said sample to a reference c-MAF expression level; andc) means for determining a therapy for preventing and/or reducing bonemetastasis in said subject based on the comparison of the quantifiedexpression level to the reference expression level. d) means forexcluding a therapy for preventing and/or reducing bone metastasis insaid subject based on the comparison of the quantified expression levelto the reference expression level.

In another aspect the invention relates to a kit comprising: i) areagent for quantifying the expression level of c-MAF in a sample of asubject suffering from prostate cancer, and ii) one or more c-MAF geneexpression level indices that have been predetermined to correlate withthe risk of bone metastasis.

Means for quantifying the expression level of c-MAF in a sample of saidsubject have been previously described in detail including 16q23 and16q22-24 locus amplification and translocation.

In a preferred embodiment, means for quantifying expression comprise aset of probes and/or primers that specifically bind and/or amplify thec-MAF gene.

In particular embodiment the prostate cancer is prostate adenoma orprostate small cell carcinoma cancer.

In particular embodiment the kit is applied, but not limited, to aprostate cancer biopsy, circulating prostate cancer cell, circulatingprostate tumor DNA.

All the particular embodiments of the methods of the present inventionare applicable to the kits of the invention and to their uses.

Method for typing a sample of a subject suffering prostate cancer.

In another aspect, the invention relates to an in vitro method fortyping a sample of a subject suffering from prostate cancer, the methodcomprising:

-   -   a) providing a sample from said subject;    -   b) quantifying the expression level of c-MAF in said sample;    -   c) typing said sample by comparing the quantified expression        level of c-MAF to a predetermined reference level of c-MAF        expression;        wherein said typing provides prognostic information related to        the risk of bone metastasis in said subject.

Means for quantifying the expression level of c-MAF in a sample of saidsubject have been previously described in detail including 16q23 and16q22-24 locus amplification and translocation.

In a preferred embodiment the sample is a tumor tissue sample, acirculating tumor cell or a circulating tumor DNA.

Method for classifying a subject suffering from prostate cancer.

In another aspect, the invention relates to a method for classifying asubject suffering from prostate cancer into a cohort, comprising: a)determining the expression level of c-MAF in a sample of said subject;b) comparing the expression level of c-MAF in said sample to apredetermined reference level of c-MAF expression; and c) classifyingsaid subject into a cohort based on said expression level of c-MAF inthe sample.

Means for quantifying the expression level of c-MAF in a sample of saidsubject have been previously described in detail including 16q23 and16q22-24 locus amplification and translocation.

In particular embodiment the prostate cancer is an adenoma or a smallcell carcinoma.

In a preferred embodiment the sample is a tumor tissue sample, acirculating tumor cell or a circulating DNA.

In a preferred embodiment said cohort comprises at least one otherindividual who has been determined to have a comparable expression levelof c-MAF in comparison to said reference expression level.

In another preferred embodiment said expression level of c-MAF in saidsample is increased relative to said predetermined reference level, andwherein the members of the cohort are classified as having increasedrisk of bone metastasis.

In another preferred embodiment said cohort is for conducting a clinicaltrial.

In a preferred embodiment, the sample is a tumor tissue sample.

EXAMPLES

Clinical relevance and prognostic value of the bone-specific metastasisgene

c-MAF was tested in a tissue micro array (TMA) including 37 Prostatetumor biopsies for which the clinical annotations of time to bonemetastasis or visceral metastasis ever was known. These tumors arerepresentative of all Prostate cancer subtypes and stages. The levels ofc-MAF were determined by immunohistochemistry (IHC) using a c-MAFspecific antibody and the association between the levels of c-MAFexpression and risk of bone relapse was established by means of Oddsratio (OR) calculations. The OR is a measure of effect size, describingthe strength of association or non-independence between two binary data.OR is described in Glas, A. S. “The diagnostic odds ratio: a singleindicator of test performance” (2003) J. of Clinical Epidemiology56:1129-1135. The Odds ratio describes the strength of association ornon-independence between two binary data values (gene of interestpositive or negative, bone metastasis positive or negative). In someembodiments, the Odds ratio is at least about 1, 1.2, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, or 5. These samples in the TMA areparaffin embedded primary tumor tissue from Prostate tumors. Thesesamples were collected at the Vall d'Hebron Oncology Institute and Valld'Hebron Hospital during regular clinical practice together with therelevant clinical data needed and the approval of the clinicalcommittee.

The samples were selected fulfill the following criteria:

5 samples belonged to patients with local disease (M0) at diagnosis witha confirmed bone relapse at any time of follow-up.

29 samples belonged to patients at diagnosis that remain metastasis freeafter at least 5 years.

The remaining 3 tumors are from patients M0 at diagnosis that latter hada relapse in any location other than bone.

Example 1 c-MAF Expression is Associated with Risk of Metastasis, inParticular Bone Metastasis Immunohistochemistry Analysis

c-MAF immunostaining was performed on TMAs. This TMA was build on glassslides and IHC was done using the Dako Link Platform according theOperating Procedure

Briefly, the immunostaining was done on 3 μm TMA tumor tissue sections,placed on positively charged glass slides (Superfrost or similar) in aDako Link platform. After deparaffinization, heat antigen retrieval wasperformed in pH 6.1, 0.01 mol/L citrate-based buffered solution (Dako).Endogenous peroxidase was quenched. The mouse polyclonal anti-c-MAFantibody (Santa Cruz) 1:100 dilution was used for 30 minutes at roomtemperature, followed by incubation with an anti-rabbit Ig dextranpolymer coupled with peroxidase (Flex+, Dako). Sections were thenvisualized with 3,3′-diaminobenzidine (DAB) and counterstained withHematoxylin.

c-MAF immunostaining was scored by a computerized algorithm. Ninerepresentative images from each specimen were acquired at 10-nmwavelength intervals between 420 and 700 nm, using a DM2000 Leicamicroscope equipped with the Nuance FX Multispectral Imaging System (CRIInc). The positive signals were converted from transmission to opticaldensity units by taking the negative log of the ratio of the sampledivided by the reference cube using a Beer law conversion. Acomputer-aided threshold was set, which created a pseudo-color imagethat highlighted all of the positive signals. Previous analysessupported the quantitative measurement of c-MAF expression.

Only the nuclei of epithelial cells (normal and malignant), but notstromal cells or lymphocytes, were automatically detected by settingdistinct size threshold and confirmed by a pathologist. For each casewas calculated the mean value of the signal intensity of all regions ofinterest for statistical analysis.

Prognostic and Predictive Value of c-MAF for Metastasis and BoneMetastasis in Prostate Cancer

The prognostic and predictive value of c-MAF expression for metastasisof Prostate cancer was evaluated. C-MAF protein levels were determinedby immunohistochemistry (IHC). MAF immunostaining was scored by acomputerized measurement. The output of the computerized measurementproduced a continuous data ranging from 1160 to 99760 optical densityunits (O.D.) for c-MAF expression. The cut off (10000 O.D.) for high anlow expression was determined based on a receiving operating curve asper standard procedures.

The results are summarized in table 3

TABLE 3 c-MAF protein expression levels Bone metastasis c-MAF expressionNO Yes <=10,000 OD 21 2   >10,000 OD 5 6

Based on this values the odds ratio of risk of suffering bone metastasisin the c-MAF high group versus the low was OR (bone metastasis at anytime)=12.60 (95% C.I. 1.93-82.09)

Based on the second cohort analyzed, we extracted some diagnosticclinical features. c-MAF high level of protein expression predicts bonemetastasis with a sensitivity of 0.75, a specificity of 0.81. This issummarized and expressed in percentages including the confidenceintervals in table 4.

TABLE 4 C.I. 95% Sensitivity 75.0% 40.9%-92.9% Specificity 80.8%62.1%-91.5%

The c-MAF gene or protein expression in Prostate tumors identifies apopulation at risk of metastasis, in particular bone metastasis at anytime.

Example 2 Gain of 16q22-24 Chromosomal Region (CNA, Copy NumberAlteration) is Associated with Risk of Bone Metastasis

We tested whether a gain in chr16q22-q24, which included c-MAF genomicloci, is associated with risk of bone metastasis in Prostate cancerpatients. To this end we used a method that identifies chr16q22-q24amplifications, in this case by means of a chr16q23 and chr14q32 dualfluorescence in situ hybridization (FISH) probe to measure the number ofcopies of the chr16q22-24 region. We also used the chr14q32 probe tonormalize tumor polyploidy.

Fluorescent in situ hybridization (FISH) analysis of 16q23, within the16q22-24, genomic region amplification, including the c-MAF gene, wasperformed on TMA above described using a fluorescence DM2000 Leicamicroscope according to the Operating Procedure. We used aSpectrumOrange probe mix that flanks the MAF gene genomic region and iscomposed of two segments that are each approximately 350 kb with anapproximately 2.2 Mb gap. The centromeric segment is located atchr16:75729985-76079705 (March 2006 assembly, UCSC Genome Browser) andthe telomeric segment is located at chr16:78290003-78635873 (March 2006assembly, UCSC Genome Browser). This probe flanks five genes VAT1L,CLEC3A, WWOX, 5srRNA and MAF (ordered from centromer to telomer).

Briefly, 16q23 region amplification, including the MAF gene, and 14q32control region, including the IgH gene, were determined by FISH on 5 μmsections of the TMA using standard procedures. Deparaffinized tissuesections were treated with 0.2 M HCl and then with sodium thiocyanate,to eliminate salt precipitates. Pretreated slides were incubated for 10min in a solution of proteinase K at 37° C. The slides were thenpostfixed in buffered formalin. The 16q23/14q32 DNA probes fluorescentlylabeled were denatured at 78° C. for 5 min and hybridized overnight at37° C. on a hotplate. Washes were performed for 2 min at 72° C. in asolution of 2×SSC/0.3% Nonidet P40. Tissue sections were counterstainedwith 10 μl of 4,6-diamino-2-phenylindole (DAPI counterstain).

Results were captured with a fluorescence DM2000 Leica microscope andanalyzed with the Nuance FX Multispectral Imaging System. FISH scoringof fluorescence signals (red for 16q23 and green for control 14q32region) were carried out by counting the number of each region copies inan average of 50 non-overlapping nuclei for each case. The prognosticand predictive value of chromosomal 16q22-24 CNA gain association withbone metastasis in Prostate cancer was evaluated. chr16q23 and chr14q32region number of copies per nuclei were determined by FISH. The expectednumber of each probe signal was two per nuclei. Amplification wasconsidered when the average 16q23 probe signals were more than more than1.5 when normalized per 14q32 region number of copies.

The results are summarized in table 5 and 6.

TABLE 5 Ratio chr16q23/chr14q32 and risk of metastasis. A tumor will bepositive for a 16q22-24 CNA gain based on a cut off >= to 1.5 copies ofthe 16q23 normalised by number of copies of the 14q32. Metastasis Ratio16q23/14q32 NO Yes <=1.5 27 2 >1.5 2 6

TABLE 6 Ratio chr16q23/chr14q32 > or = 1.5 and prediction of risk ofbone metastasis. A tumor will be positive for a chr16q22-24 CNA gainbased on a cut off >= to 1.5 copies of the chr16q23 normalized by numberof copies of the chr14q32. Bone metastasis Ratio 16q23/14q32 NO Yes<=1.5 31 2 >1.5 1 3

Based on these values we calculated the odds ratio of risk of sufferingmetastasis and bone metastasis in the chr16q22-24 gain or CNA gainpositive group versus the negative. Based on this estimation, the OR forthe chr16q22-24 CNA positive patients to suffer a metastasis was 40.50(95% CI 4.72-347.82), and the OR for the chr16q22-24 CNA gain normalizedusing 14q32 positive patients versus the control and bone metastasis was46.50 (95% CI 3.20-676.24). The small size of the cohort made theestimates imprecise but within a clinically relevant OR of least 4.72and 3.20 with a 95% confidence in each case.

Based on the data analyzed by FISH, we extracted some diagnosticclinical features. Chr16q22-24 CNA gain (>=1.5 16q23 copies per cellnormalized to chr14q32) predicts Prostate cancer risk of metastasis witha sensitivity of 0.75, a specificity of 0.93. This results expressed inpercentages are summarized as follows in table 7 including 95%confidence intervals (C.I.).

TABLE 7 Diagnostic clinical features based on Chr16q22-24 CNA gain(>=1.5 16q23 copies per cell normalized to chr14q32) for the predictionof prostate cancer risk of metastasis C.I. 95% Sensitivity 75.0%40.9%-92.9% Specificity 93.1% 78.0%-98.1%

Based on the data analyzed by FISH, we extracted some diagnosticclinical features. Chr16q22-24 CNA gain (>=1.5 16q23 copies per cellnormalized to chr14q32) predicts Prostate cancer risk of bone metastasiswith a sensitivity of 0.60, a specificity of 0.97. This resultsexpressed in percentages are summarized as follows in table 8 including95% confidence intervals (C.I.)

TABLE 8 Diagnostic clinical features based on Chr16q22-24 CNA gain(>=1.5 16q23 copies per cell normalized to chr14q32) for the predictionof prostate cancer risk of bone metastasis C.I. 95% Sensitivity 60.0%23.1%-88.2% Specificity 96.9% 84.3%-99.4%

The chr16q22-24, and in particularly chr16q23, CNA gain in Prostatetumors strongly predicts and is associated with risk of metastasis andbone metastasis at any time.

1. An in vitro method for the diagnosis of metastasis in a subject withprostate cancer and/or for the prognosis of the tendency to developmetastasis in a subject with prostate cancer, said method comprising:(i) quantifying the c-MAF gene expression level in a prostate tumorsample of said subject and (ii) comparing the expression level obtainedin (i) with the expression level of the c-MAF gene in a control sample,wherein if the expression level of the c-MAF gene in said tumor sampleis increased with respect to the expression level of the c-MAF gene inthe control sample, then said subject has a positive diagnosis formetastasis or a greater tendency to develop metastasis.
 2. An in vitromethod for designing a customized therapy for a subject with prostatecancer which comprises (i) quantifying the c-MAF gene expression levelin a prostate tumor sample of said subject, and (ii) comparing theexpression level obtained in (i) with the expression level of the c-MAFgene in a control sample, wherein if the expression level of the c-MAFgene in the tumor sample is increased with respect to the expressionlevel of the c-MAF gene in the control sample, then said subject issusceptible to receive a therapy intended to prevent, inhibit and/ortreat metastasis of the cancer.
 3. The method according to claim 1 or 2,wherein the metastasis is bone metastasis.
 4. The method according toclaim 3, wherein the bone metastasis is osteolytic metastasis.
 5. An invitro method for designing a customized therapy for a subject havingprostate cancer with bone metastasis which comprises (i) quantifying thec-MAF gene expression level in a bone metastatic tumor tissue sample ofsaid subject, and (ii) comparing the expression level obtained in step(i) with the expression level of the c-MAF gene in a control sample,wherein if the c-MAF gene expression level in the tumor tissue sample isincreased with respect to the expression level of the c-MAF gene in thecontrol sample, then said subject is susceptible to receive a therapyintended to prevent or inhibit bone degradation.
 6. The method accordingto claim 5, wherein the agent intended to prevent or inhibit bonedegradation is selected from the group consisting of: a bisphosphonate,a RANKL inhibitor, PTH, a PTHLH inhibitor (including neutralizingantibodies and peptides), a PRG analog, strontium ranelate, a DKK-1inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, calcitonin, Radium-223, a CCR5 antagonist, a Src kinaseinhibitor, a COX-2 inhibitor, an mTor inhibitor, and a cathepsin Kinhibitor.
 7. The method according to claim 6, wherein the RANKLinhibitor is selected from the group consisting of: a RANKL specificantibody, a RANKL-specific nanobody, and osteoprotegerin.
 8. The methodaccording to claim 7, wherein the RANKL specific antibody is denosumab.9. The method according to claim 6, wherein the bisphosphonate iszoledronic acid.
 10. The method according to any of claims 1 to 9,wherein the quantification of the c-MAF gene expression level comprisesquantifying the messenger RNA (mRNA) of said gene, or a fragment of saidmRNA, the complementary DNA (cDNA) of said gene, or a fragment of saidcDNA.
 11. The method according to claim 10, wherein the expression levelis quantified by means of a quantitative polymerase chain reaction (PCR)or a DNA or RNA array or nucleotide hybridization technique.
 12. Themethod according to any of claims 1 to 10, wherein the quantification ofthe c-MAF gene expression level comprises quantifying the level ofprotein encoded by said gene or of a variant thereof.
 13. The methodaccording to claim 12, wherein the level of protein is quantified bymeans of western blot, ELISA, immunohistochemistry or a protein array.14. An in vitro method for diagnosing metastasis in a subject withprostate cancer and/or for the prognosis of the tendency to developmetastasis in a subject with prostate cancer which comprises determiningif the c-MAF gene is amplified in a tumor sample of said subjectrelative to a reference gene copy number, wherein an amplification ofthe c-MAF gene with respect to said reference gene copy number isindicative of the presence of metastasis or an increased risk ofdeveloping metastasis.
 15. The method according to claim 14, wherein theamplification of the c-MAF gene is determined by means of determiningthe amplification of the locus 16q22-q24.
 16. The method according toclaim 14 or 15, wherein the amplification of the c-MAF gene isdetermined by means of using a c-MAF gene-specific probe.
 17. The methodaccording to any of claims 14-16, wherein the reference gene copy numberis that of a tumor tissue sample of prostate cancer from a subject whohas not suffered metastasis.
 18. The method according to any of claims14-17, wherein the amplification is determined by means of in situhybridization or PCR.
 19. The method according to any of claims 14-18,wherein the metastasis is bone metastasis.
 20. The method according toclaim 19, wherein the bone metastasis is osteolytic metastasis.
 21. Useof a c-MAF inhibitory agent in the preparation of a medicinal productfor treating and/or preventing bone metastasis from prostate cancer. 22.The use according to claim 21, wherein the c-MAF inhibitory agent isselected from the group consisting of: a c-MAF specific siRNA, a c-MAFspecific antisense oligonucleotide, a c-MAF specific ribozyme, a c-MAFinhibitory antibody or nanobody, a dominant negative c-MAF variant, acompound from Table 1 or from Table 2, a c-MAF specific small molecule,a c-MAF specific antibody, a c-MAF specific antibody-like molecule, ac-MAF specific structurally constrained (cyclical) peptide, a c-MAFspecific stapled peptide, or a c-MAF specific alphabody.
 23. Use of anagent capable of preventing or inhibiting bone degradation in thepreparation of a medicinal product for the treatment of bone metastasisin a subject suffering prostate cancer and having elevated c-MAF levelsin a metastatic tumor sample with respect to a control sample.
 24. Useaccording to claim 23, wherein the agent capable of avoiding orpreventing bone degradation is selected from the group consisting of: abisphosphonate, a RANKL inhibitor, PTH, PTHLH inhibitor (includingneutralizing antibodies and peptides), a PRG analog, strontium ranelate,a DKK-1 inhibitor, a dual MET and VEGFR2 inhibitor, an estrogen receptormodulator, an EGFR inhibitor, calcitonin, Radium-223, a CCR5 antagonist,a Src kinase inhibitor, a COX-2 inhibitor, an mTor inhibitor, and acathepsin K inhibitor.
 25. Use according to claim 25, wherein the RANKLinhibitor is selected from the group of: a RANKL specific antibody, aRANKL specific nanobody, and osteoprotegerin.
 26. Use according to claim26, wherein the RANKL specific antibody is denosumab.
 27. Use accordingto claim 24, wherein the bisphosphonate is zoledronic acid.
 28. Useaccording to any of claims 24-27, wherein the bone metastasis isosteolytic metastasis.
 29. A kit for predicting bone metastasis of aprostate cancer in a subject suffering from said cancer, the kitcomprising: a) means for quantifying the expression level of c-MAF in atumor sample of said subject; and b) means for comparing the quantifiedlevel of expression of c-MAF in said sample to a reference c-MAFexpression level.
 30. An in vitro method for typing a sample of asubject suffering from prostate cancer, the method comprising: a)providing a sample from said subject; b) quantifying the expressionlevel of c-MAF in said sample; c) typing said sample by comparing thequantified expression level of c-MAF to a predetermined reference levelof c-MAF expression; wherein said typing provides prognostic informationrelated to the risk of bone metastasis in said subject.
 31. A method forpreventing, inhibiting or reducing the risk of bone metastasis in asubject suffering from prostate cancer, said method comprisingadministering to said subject an agent that prevents or reduces bonemetastasis, wherein said agent is administered in accordance with atreatment regimen determined from quantifying the expression level ofc-MAF in said subject.
 32. A method of classifying a subject sufferingfrom prostate cancer into a cohort, comprising: a) determining theexpression level of c-MAF in a prostate tumor sample of said subject; b)comparing the expression level of c-MAF in said sample to apredetermined reference level of c-MAF expression; and c) classifyingsaid subject into a cohort based on said expression level of c-MAF inthe sample.
 33. The method according to claim 6, wherein the RANKLspecific nanobody is ALX-0141.
 34. The method according to claim 6,wherein the dual MET and VEGFR2 inhibitor is Cabozantinib.
 35. The useaccording to claim 24, wherein the RANKL specific nanobody is ALX-9141.36. The use according to claim 24, wherein the dual MET and VEGFR2inhibitor is Cabozantinib.
 37. The method according to claim 16, whereinthe c-MAF gene-specific probe is Vysis LSI/IGH MAF Dual Color DualFusion Probe.
 38. A kit for determining a therapy for a subjectsuffering from prostate cancer, the kit comprising: a) means forquantifying the expression level of c-MAF in a tumor sample of saidsubject; b) means for comparing the quantified expression level of c-MAFin said sample to a reference c-MAF expression level; and c) means fordetermining a therapy for preventing, inhibiting and/or reducing bonemetastasis in said subject based on the comparison of the quantifiedexpression level to the reference expression level.
 39. A kitcomprising: i) a reagent for quantifying the expression level of c-MAFin a tumor sample of a subject suffering from prostate cancer, and ii)one or more c-MAF gene expression level indices that have beenpredetermined to correlate with the risk of bone metastasis.
 40. A kitaccording to claim 38-39 wherein said means for quantifying expressioncomprise a set of probes and/or primers that specifically bind and/oramplify the c-MAF gene, the 16q23 locus or the 16q22-16q24 chromosomalregion.
 41. An in vitro method for typing a sample of a subjectsuffering from prostate cancer, the method comprising: (i) providing atumor sample from said subject; (ii) quantifying the expression level ofc-MAF in said sample; (iii) typing said sample by comparing thequantified expression level of c-MAF to a predetermined reference levelof c-MAF expression; wherein said typing provides prognostic informationrelated to the risk of bone metastasis in said subject.
 42. A method forpreventing, inhibiting, or reducing the risk of bone metastasis in asubject suffering from prostate cancer, said method comprisingadministering or not to said subject an agent that prevents or reducesbone metastasis, wherein said agent is administered in accordance with atreatment regimen determined at least in part from quantifying theexpression level of c-MAF in said subject.
 43. A method of classifying asubject suffering from prostate cancer into a cohort, comprising: a)determining the expression level of c-MAF in a cancer tumor sample ofsaid subject; b) comparing the expression level of c-MAF in said sampleto a predetermined reference level of c-MAF expression; and c)classifying said subject into a cohort based on said expression level ofc-MAF in said sample.
 44. A method according to claim 43, wherein saidcohort comprises at least one other individual who has been determinedto have a comparable expression level of c-MAF in comparison to saidreference expression level.
 45. A method according to claim 43 or 44,wherein said expression level of c-MAF in said sample is increasedrelative to said predetermined reference level, and wherein members ofthe cohort are classified as having increased risk of bone metastasis.46. A method according to any of claims 43-45, wherein the cohort is forconducting a clinical trial.
 47. An in vitro method for predicting bonemetastasis of prostate cancer in a subject suffering said cancer, saidmethod comprising determining if the c-MAF gene is translocated in atumor sample of said subject, wherein translocation of the c-MAF gene isindicative of an increased risk of bone metastasis.
 48. An in vitromethod for designing a customized therapy for a subject having prostatecancer with bone metastasis which comprises determining if the c-MAFgene is amplified in a tumor sample of said subject relative to areference gene copy number, wherein an amplification of the c-MAF genewith respect to said reference gene copy number indicates that thesubject is a candidate for receiving a therapy intended to prevent orinhibit bone degradation.
 49. The method according to claim 48, whereinthe agent intended to prevent or inhibit bone degradation is selectedfrom the group consisting of: a bisphosphonate, a RANKL inhibitor, PTH,a PTHLH inhibitor (including neutralizing antibodies and peptides), aPRG analog, strontium ranelate, a DKK-1 inhibitor, a dual MET and VEGFRinhibitor, an estrogen receptor modulator, calcitonin, Radium-223, aCCR5 antagonist, a Src kinase inhibitor, a COX-2 inhibitor, an mTorinhibitor, and a cathepsin K inhibitor.
 50. The method according toclaim 49, wherein the RANKL inhibitor is selected from the groupconsisting of: a RANKL specific antibody, a RANKL-specific nanobody, andosteoprotegerin.
 51. The method according to claim 50, wherein the RANKLspecific antibody is denosumab.
 52. The method according to claim 49,wherein the bisphosphonate is zoledronic acid.
 53. The method accordingto claims 23-28, wherein said means for quantifying expression comprisea set of probes and/or primers that specifically bind and/or amplify thec-MAF gene, the 16q23 locus or the 16q22-16q24 chromosomal region. 54.The method according to claims 29-32, wherein said means for quantifyingexpression comprise a set of probes and/or primers that specificallybind and/or amplify the c-MAF gene, the 16q23 locus or the 16q22-16q24chromosomal region.
 55. The method according to claims 41-47, whereinsaid means for quantifying expression comprise a set of probes and/orprimers that specifically bind and/or amplify the c-MAF gene, the 16q23locus or the 16q22-16q24 chromosomal region.